Sample records for lysozyme protein solutions

  1. Lysozyme Protein Solution with an Intermediate Range Order Structure

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    Liu, Yun [National Institute of Standards and Technology (NIST); Porcar, L. [National Institute of Standards and Technology (NIST); Chen, Wei-Ren [ORNL; Chen, Jinhong [Memorial Sloan-Kettering Cancer Center; Falus, Peter [ORNL; Fratini, Emiliano [University of Florence; Faraone, Antonio [National Institute of Standards and Technology (NIST); Baglioni, P [University of Florence


    The formation of equilibrium clusters has been studied in both a prototypical colloidal system and protein solutions. The appearance of a low-Q correlation peak in small angle scattering patterns of lysozyme solution was attributed to the cluster-cluster correlation. Consequently, the presence of long-lived clusters has been established. By quantitatively analyzing both the SANS (small angle neutron scattering) and NSE (neutron spin echo) data of lysozyme solution using statistical mechanics models, we conclusively show in this paper that the appearance of a low-Q peak is not a signature of the formation of clusters. Rather, it is due to the formation of an intermediate range order structure governed by a short-range attraction and a long-range repulsion. We have further studied dynamic features of a sample with high enough concentration at which clusters are formed in solution. From the estimation of the mean square displacement by using short-time and long-time diffusion coefficient measured by NSE and NMR, we find that these clusters are not permanent but have a finite lifetime longer than the time required to diffuse over a distance of a monomer diameter.

  2. Small-angle neutron scattering study of protein crowding in liquid and solid phases: lysozyme in aqueous solution, frozen solution, and carbohydrate powders. (United States)

    Curtis, Joseph E; Nanda, Hirsh; Khodadadi, Sheila; Cicerone, Marcus; Lee, Hyo Jin; McAuley, Arnold; Krueger, Susan


    The structure, interactions, and interprotein configurations of the protein lysozyme were studied in a variety of phases. These properties have been studied under a variety of solution conditions before, during, and after freezing and after freeze-drying in the presence of glucose and trehalose. Contrast variation experiments have also been performed to determine which features of the scattering in the frozen solutions are from the protein and which are from the ice structure. Data from lysozyme at concentrations ranging from 1 to 100 mg/mL in solution and water ice with NaCl concentrations ranging from 0 to 0.4 mol/L are fit to model small-angle neutron scattering (SANS) intensity functions consisting of an ellipsoidal form factor and either a screened-Coulomb or hard-sphere structure factor. Parameters such as protein volume fraction and long dimension are followed as a function of temperature and salt concentration. The SANS results are compared to real space models of concentrated lysozyme solutions at the same volume fractions obtained from Monte Carlo simulations. A cartoon representation of the frozen lysozyme solution in 0 mol/L NaCl is presented based on the SANS and Monte Carlo results, along with those obtained from other complementary methods.

  3. Characteristics of hydration water around hen egg lysozyme as the protein model in aqueous solution. FTIR spectroscopy and molecular dynamics simulation. (United States)

    Panuszko, Aneta; Wojciechowski, Marek; Bruździak, Piotr; Rakowska, Paulina W; Stangret, Janusz


    In this paper, the hydration of a model protein--hen egg white lysozyme in aqueous solution has been presented. The leading method used was FTIR spectroscopy with an application of a technique of semi-heavy water (HDO) isotope dilution. Analysis of spectra of HDO isotopically diluted in water solution of lysozyme allowed us to isolate HDO spectra affected by lysozyme, and thus to characterise the energetic state of water molecules and their arrangement around protein molecules. The number of water molecules and the shape of the affected HDO spectrum were obtained using a classical and a chemometric method. This shape showed that the HDO spectrum affected by lysozyme may be presented as a superposition of two spectra corresponding to HDO affected by N-methylacetamide and the carboxylate anion (of the formic acid). Moreover, based on the difference in intermolecular distances distribution of water molecules (obtained from spectral data), we demonstrated that the lysozyme molecule causes a decrease in population of weak hydrogen bonds, and concurrently increases the probability of an occurrence of short hydrogen bonds in water affected by lysozyme. This conclusion was also confirmed by the molecular dynamics (MD) simulation.

  4. Terahertz absorption of lysozyme in solution (United States)

    Martin, Daniel R.; Matyushov, Dmitry V.


    Absorption of radiation by solution is described by its frequency-dependent dielectric function and can be viewed as a specific application of the dielectric theory of solutions. For ideal solutions, the dielectric boundary-value problem separates the polar response into the polarization of the void in the liquid, created by the solute, and the response of the solute dipole. In the case of a protein as a solute, protein nuclear dynamics do not project on significant fluctuations of the dipole moment in the terahertz domain of frequencies and the protein dipole can be viewed as dynamically frozen. Absorption of radiation then reflects the interfacial polarization. Here we apply an analytical theory and computer simulations to absorption of radiation by an ideal solution of lysozyme. Comparison with the experiment shows that Maxwell electrostatics fails to describe the polarization of the protein-water interface and the "Lorentz void," which does not anticipate polarization of the interface by the external field (no surface charges), better represents the data. An analytical theory for the slope of the solution absorption against the volume fraction of the solute is formulated in terms of the cavity field response function. It is calculated from molecular dynamics simulations in good agreement with the experiment. The protein hydration shell emerges as a separate sub-ensemble, which, collectively, is not described by the standard electrostatics of dielectrics.

  5. Salt-specific effects in lysozyme solutions

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    T. Janc


    Full Text Available The effects of additions of low-molecular-mass salts on the properties of aqueous lysozyme solutions are examined by using the cloud-point temperature, T_{cloud}, measurements. Mixtures of protein, buffer, and simple salt in water are studied at pH=6.8 (phosphate buffer and pH=4.6 (acetate buffer. We show that an addition of buffer in the amount above I_{buffer} = 0.6 mol dm^{-3} does not affect the T_{cloud} values. However, by replacing a certain amount of the buffer electrolyte by another salt, keeping the total ionic strength constant, we can significantly change the cloud-point temperature. All the salts de-stabilize the solution and the magnitude of the effect depends on the nature of the salt. Experimental results are analyzed within the framework of the one-component model, which treats the protein-protein interaction as highly directional and of short-range. We use this approach to predict the second virial coefficients, and liquid-liquid phase diagrams under conditions, where T_{cloud} is determined experimentally.

  6. Reentrant condensation of lysozyme: Implications for studying dynamics of lysozyme in aqueous solutions of lithium chloride

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    Mamontov, Eugene [ORNL; O' Neill, Hugh Michael [ORNL


    Recent studies have outlined the use of eutectic solution of lithium chloride in water to study microscopic dynamics of lysozyme in an aqueous solvent that is remarkably similar to pure water in many respects, yet allows experiments over a wide temperature range without the solvent crystallization. The eutectic point in (H2O)R(LiCl) system corresponds to R 7.3, and it is of interest to investigate whether less concentrated aqueous solutions of LiCl could be employed in low-temperature studies of a solvated protein. We have investigated a range of concentrations of lysozyme and LiCl in aqueous solutions to identify systems that do not show phase separation and avoid solvent crystallization on cooling down. Compared to the lysozyme concentration in solution, the concentration of LiCl in the aqueous solvent plays the major role in determining systems suitable for low-temperature studies. We have observed interesting and rich phase behavior reminiscent of reentrant condensation of proteins.

  7. Protein crowding in solution, frozen and freeze-dried states: small-angle neutron and X-ray scattering study of lysozyme/sorbitol/water systems (United States)

    Krueger, Susan; Khodadadi, Sheila; Clark, Nicholas; McAuley, Arnold; Cristiglio, Viviana; Theyencheri, Narayanan; Curtis, Joseph; Shalaev, Evgenyi


    For effective preservation, proteins are often stored as frozen solutions or in glassy states using a freeze-drying process. However, aggregation is often observed after freeze-thaw or reconstitution of freeze-dried powder and the stability of the protein is no longer assured. In this study, small-angle neutron and X-ray scattering (SANS and SAXS) have been used to investigate changes in protein-protein interaction distances of a model protein/cryoprotectant system of lysozyme/sorbitol/water, under representative pharmaceutical processing conditions. The results demonstrate the utility of SAXS and SANS methods to monitor protein crowding at different stages of freezing and drying. The SANS measurements of solution samples showed at least one protein interaction peak corresponding to an interaction distance of ~ 90 Å. In the frozen state, two protein interaction peaks were observed by SANS with corresponding interaction distances at 40 Å as well as 90 Å. On the other hand, both SAXS and SANS data for freeze-dried samples showed three peaks, suggesting interaction distances ranging from ~ 15 Å to 170 Å. Possible interpretations of these interaction peaks will be discussed, as well as the role of sorbitol as a cryoprotectant during the freezing and drying process.

  8. Comparative evaluation of multi-purpose solutions in the stabilization of tear lysozyme. (United States)

    Barniak, Vicki L; Burke, Susan E; Venkatesh, Srini


    The range and extent of tear proteins removed by various multi-purpose solutions has been investigated, but there is little information in the literature about their ability to prevent denaturation of tear proteins, particularly lysozyme. The purpose of this study was to determine the ability of Bausch+Lomb Biotrue™ multi-purpose solution and other care solutions to affect denaturation of lysozyme using a lysozyme activity assay. The test solutions used were: Biotrue multi-purpose solution, Bausch+Lomb renu(®) fresh™, formerly ReNu MultiPlus(®), Alcon OPTI-FREE RepleniSH, Alcon OPTI-FREE EXPRESS, CIBA VISION AQuify, and AMO COMPLETE Multi-Purpose Solution Easy Rub Formula. A phosphate-buffered saline (PBS) solution served as a control. The test and control solutions containing lysozyme were exposed to sodium dodecyl sulfate (SDS), a known denaturant of the enzyme. The assay was based on digestion of the cell wall of Micrococcus luteus in a suspension, a substrate sensitive to active lysozyme. Enzymatic activity against M. luteus was used to assess activity of lysozyme. The decrease in the turbidity of the cell wall suspension, a measure of relative enzyme activity, was determined by following the decrease in absorbance (at 450nm) over time using a spectrophotometer. Statistically significant greater stabilization of lysozyme was observed with Biotrue multi-purpose solution and renu fresh than with OPTI-FREE RepleniSH, OPTI-FREE EXPRESS, AQuify, COMPLETE Multi-Purpose Solution Easy Rub Formula, and a PBS control. The lysozyme activity assay revealed that Biotrue multi-purpose solution and renu fresh have the ability to stabilize lysozyme under conditions that typically denature the protein.

  9. Study of the interactions between lysozyme and a fully-fluorinated surfactant in aqueous solution at different surfactant-protein ratios. (United States)

    Ruso, Juan M; González-Pérez, Alfredo; Prieto, Gerardo; Sarmiento, Félix


    The interactions of a fluorinated surfactant, sodium perfluorooctanoate, with lysozyme, have been investigated by a combination of UV absorbance, electrical conductivity and dynamic light scattering to detect and to characterize the conformational transitions of lysozyme. By using difference spectroscopy, the transition was followed as a function of surfactant concentration, and the data were analyzed to obtain the Gibbs energy of the transition in water (DeltaGw(o)) and in a hydrophobic environment (DeltaGh(o)) for saturated protein-surfactant complexes. Electrical conductivity was used to determine the critical micelle concentration of the surfactant in the presence of different lysozyme concentration. From these results, the average number of surfactant monomer per protein molecule was calculated. Finally, dynamic light scattering show that only changes in the secondary structure of the protein can be observed.

  10. Stability of lysozyme in aqueous extremolyte solutions during heat shock and accelerated thermal conditions.

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    Christina Avanti

    Full Text Available The purpose of this study was to investigate the stability of lysozyme in aqueous solutions in the presence of various extremolytes (betaine, hydroxyectoine, trehalose, ectoine, and firoin under different stress conditions. The stability of lysozyme was determined by Nile red Fluorescence Spectroscopy and a bioactivity assay. During heat shock (10 min at 70°C, betaine, trehalose, ectoin and firoin protected lysozyme against inactivation while hydroxyectoine, did not have a significant effect. During accelerated thermal conditions (4 weeks at 55°C, firoin also acted as a stabilizer. In contrast, betaine, hydroxyectoine, trehalose and ectoine destabilized lysozyme under this condition. These findings surprisingly indicate that some extremolytes can stabilize a protein under certain stress conditions but destabilize the same protein under other stress conditions. Therefore it is suggested that for the screening extremolytes to be used for protein stabilization, an appropriate storage conditions should also be taken into account.

  11. Impact of a Reducing Agent on the Dynamic Surface Properties of Lysozyme Solutions. (United States)

    Tihonov, Michael M; Kim, Viktoria V; Noskov, Boris A


    Disulfide bond shuffling in the presence of the reducing agents dithiothreitol (DTT) or β-mercaptoethanol (BME) strongly affects the surface properties of lysozyme solutions. The addition of 0.32 mM DTT substantially alters the kinetic dependencies of the dynamic surface elasticity and surface tension relative to those of pure protein solutions. The significant increase in the dynamic surface elasticity likely relates to the cross-linking between lysozyme molecules and the formation of a dense layer of protein globules stabilized by intermolecular disulfide bonds at the liquid/gas interface. This effect differs from the previously described influence of chaotropic denaturants, such as guanidine hydrochloride (GuHCl) and urea, on the surface properties of lysozyme solutions. If both chaotropic and reducing agents are added to protein solutions simultaneously, their effects become superimposed. In the case of mixed lysozyme/GuHCl/DTT solutions, the dynamic surface elasticity near equilibrium decreases as the GuHCl concentration increases because of the gradual loosening of the cross-linked layer of protein globules but remains much higher than that of lysozyme/GuHCl solutions.

  12. Lysozymes and lysozyme-like proteins from the fall armyworm, Spodoptera frugiperda. (United States)

    Chapelle, Michael; Girard, Pierre-Alain; Cousserans, François; Volkoff, Nathalie-Anne; Duvic, Bernard


    Lysozyme is an important component of the insect non-specific immune response against bacteria that is characterized by its ability to break down bacterial cell-walls. By searching an EST database from the fall armyworm, Spodoptera frugiperda (Negre et al., 2006), we identified five sequences encoding proteins of the lysozyme family. The deduced protein sequences corresponded to three classical c-type lysozymes Sf-Lys1, Sf-Lys2 and Sf-Lys3, and two lysozyme-like proteins, Sf-LLP1 and Sf-LLP2. Sf-Lys1 was purified from the hemolymph of Escherichia coli-challenged S. frugiperda larvae. The mature protein had a molecular mass of 13.975 Da with an isoelectric point of 8.77 and showed 98.3% and 96.7% identity with lysozymes from Spodoptera litura and Spodoptera exigua, respectively. As the other insect lysozymes, Sf-Lys1 was active against gram positive bacteria such as Micrococcus luteus but also induced a slight permeabilization of the inner membrane of E. coli. Genes encoding these five Sf-Lys or Sf-LLPs were differentially up-regulated in three immune-competent tissues (hemocytes, fat body and gut) after challenges with non-pathogenic bacteria, E. coli and M. luteus, or entomopathogenic bacterium, Photorhabdus luminescens. Sf-Lys1 and Sf-Lys2 were mainly induced in fat body in the presence of E. coli or P. luminescens. Sf-Lys3, which had an acidic isoelectric point, was found to be the most up-regulated of all five Sf-Lys or Sf-LLPs in hemocytes and gut after challenge with P. luminescens. More molecular data are now available to investigate differences in physiological functions of these different members of the lysozyme superfamily.

  13. Laser ablation of the protein lysozyme

    DEFF Research Database (Denmark)

    Schou, Jørgen; Canulescu, Stela; Amoruso, Salvatore

    Lysozyme is a well-known protein, which is used in food processing because of its bactericidal properties. The mass (14307 amu) is in the range in which it easily can be monitored by mass spectrometric methods, for example by MALDI (Matrix assisted laser desorption ionization). We have recently...... produced thin films of average thickness up to 300 nm, which not only contained a significant amount of intact molecules, but also maintained the bioactivity. These films were produced by a nanosecond laser in the UV regime at 355 nm with 2 J/cm2. The surprising fact that these molecules can be transferred...... to a substrate as intact molecules by the violent laser impact ( up to 50 mJ/pulse) has not yet been understood. One issue is that up to 150 ng/pulse is removed by the laser, and much of the material is ejected from the target in relatively large chunks. We have explored as well the excitation mechanics by laser...

  14. Tear Lipids Interfacial Rheology: Effect of Lysozyme and Lens Care Solutions (United States)

    Svitova, Tatyana F.; Lin, Meng C.


    Purpose To evaluate the interfacial properties of ex vivo tear lipid multilayers with controlled and varying thickness. The influence of lysozyme and surfactant-containing multi-purpose lens care solutions (MPS) on interfacial rheology of lipids and mixed lipid-protein films were studied. Methods Lipids were extracted from lotrafilcon A lenses worn continuously for 1 month. Interfacial properties of the lipids without and with lysozyme in the aqueous phase were examined using tensiometry and step-strain relaxation. Lipid-lysozyme multilayers were exposed to either diluted Optifree Express (OFX) or Optifree Replenish (OFR) for 30 min, and then MPS was displaced from the bulk phase. Surface tension and rheological parameters before and after MPS exposure were measured and compared. Results Thick lipid films exerted high surface pressure at the air-aqueous interface, 50 ± 2 mN/m, with little inter- and intra-subject variability. The rheological storage modulus (E∞; 25.3 ± 2 mN/m) and relaxation time (τ; 87 ± 25 s) were similar among subjects. Neither lysozyme nor MPS changed the surface tension of the lipid multilayers. Lysozyme adsorbed irreversibly onto multilayers without changing E∞, but increased τ 2.5 times. Exposure of mixed multilayers to OFX reduced E∞ to less than a half of its original value (13 ± 4.5 mN/m; p rheology of the ex vivo lipids. OFX and OFR changed rheological properties of the mixed films to different extents. PMID:19901859

  15. Conductivity and Viscosity Measurements for Binary Lysozyme Chloride Aqueous Solution and Ternary Lysozyme-Salt-Water Solution

    CERN Document Server

    Buzatu, D; Buzatu, F D


    We use the conductimetric method, adequate to electrolytes, to determine the lysozyme charge in lys-water and ternary lys-salt-water systems. We measured also the viscosities for the above binary and ternary systems in the same conditions at pH$=4.5$ and T$=298$ K, measurements that allow us to see any effect of viscosity on cations mobilities and implicitly on the lysozyme charge. The method is illustrated for the lysozyme chloride aqueous solution system at 25$^o$ C, using the data reported here for pH$=4.5$ at 0.15, 0.6, 0.8, 1., 1.5, 2., 2.5, 3., 3.5 mM (mg/mL) lysozyme chloride concentrations. The method was also applied to ternary lys-salt-water systems in the same conditions at pH$=4.5$ and T$=25^o$ C. Ternary conductivities are reported for a mean concentration 0.6 mM of lysozyme chloride in all systems and a mean concentration 0.01, 0.025, 0.05, 0.1, 0.175, 0.2, 0.5, 0.7, 0.9, 1.2, 1.3 and 1.4 M for NaCl; 0.005, 0.01, 0.05, 0.1, 0.175, 0.2, 0.5, 0.7, 0.9, 1.2, 1.3, 1.4 and 1.5 M for KCl; 0.005, 0.01,...

  16. Pulsed electric field (PEF)-induced aggregation between lysozyme, ovalbumin and ovotransferrin in multi-protein system. (United States)

    Wu, Li; Zhao, Wei; Yang, Ruijin; Yan, Wenxu


    The aggregation of multi-proteins is of great interest in food processing and a good understanding of the formation of aggregates during PEF processing is needed for the application of the process to pasteurize protein-based foods. The aggregates formation of a multi-protein system (containing ovalbumin, ovotransferrin and lysozyme) was studied through turbidity, size exclusion chromatography and SDS-PAGE patterns for interaction studies and binding forces. Results from size exclusion chromatography indicated that there was no soluble aggregates formed during PEF processing. The existence of lysozyme was important to form insoluble aggregates in the chosen ovalbumin solution. The results of SDS-PAGE patterns indicated that lysozyme was prone to precipitate, and was relatively the higher component of aggregates. Citric acid could be effective in inhibiting lysozyme from interacting with other proteins during PEF processing. Blocking the free sulphydryl by N-ethylmaleimide (NEM) did not affect aggregation inhibition.

  17. Stability of lysozyme in aqueous extremolyte solutions during heat shock and accelerated thermal conditions

    NARCIS (Netherlands)

    Avanti, Christina; Saluja, Vinay; Van Streun, Erwin L. P.; Frijlink, Henderik W.; Hinrichs, Wouter L. J.


    The purpose of this study was to investigate the stability of lysozyme in aqueous solutions in the presence of various extremolytes (betaine, hydroxyectoine, trehalose, ectoine, and firoin) under different stress conditions. The stability of lysozyme was determined by Nile red Fluorescence Spectrosc

  18. Protein aggregation in salt solutions (United States)

    Kastelic, Miha; Kalyuzhnyi, Yurij V.; Hribar-Lee, Barbara; Dill, Ken A.; Vlachy, Vojko


    Protein aggregation is broadly important in diseases and in formulations of biological drugs. Here, we develop a theoretical model for reversible protein–protein aggregation in salt solutions. We treat proteins as hard spheres having square-well-energy binding sites, using Wertheim’s thermodynamic perturbation theory. The necessary condition required for such modeling to be realistic is that proteins in solution during the experiment remain in their compact form. Within this limitation our model gives accurate liquid–liquid coexistence curves for lysozyme and γ IIIa-crystallin solutions in respective buffers. It provides good fits to the cloud-point curves of lysozyme in buffer–salt mixtures as a function of the type and concentration of salt. It than predicts full coexistence curves, osmotic compressibilities, and second virial coefficients under such conditions. This treatment may also be relevant to protein crystallization. PMID:25964322

  19. Structural basis of protein oxidation resistance: a lysozyme study.

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    Marion Girod

    Full Text Available Accumulation of oxidative damage in proteins correlates with aging since it can cause irreversible and progressive degeneration of almost all cellular functions. Apparently, native protein structures have evolved intrinsic resistance to oxidation since perfectly folded proteins are, by large most robust. Here we explore the structural basis of protein resistance to radiation-induced oxidation using chicken egg white lysozyme in the native and misfolded form. We study the differential resistance to oxidative damage of six different parts of native and misfolded lysozyme by a targeted tandem/mass spectrometry approach of its tryptic fragments. The decay of the amount of each lysozyme fragment with increasing radiation dose is found to be a two steps process, characterized by a double exponential evolution of their amounts: the first one can be largely attributed to oxidation of specific amino acids, while the second one corresponds to further degradation of the protein. By correlating these results to the structural parameters computed from molecular dynamics (MD simulations, we find the protein parts with increased root-mean-square deviation (RMSD to be more susceptible to modifications. In addition, involvement of amino acid side-chains in hydrogen bonds has a protective effect against oxidation Increased exposure to solvent of individual amino acid side chains correlates with high susceptibility to oxidative and other modifications like side chain fragmentation. Generally, while none of the structural parameters alone can account for the fate of peptides during radiation, together they provide an insight into the relationship between protein structure and susceptibility to oxidation.

  20. Dimer formation in radiation-irradiated aqueous solution of lysozyme studied by light-scattering-intensity measurement

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    Hashimoto, S.; Masuda, T.; Kondo, M. (Institute of Physical and Chemical Research, Wako, Saitama (Japan); Tokyo Metropolitan Univ. (Japan). Faculty of Science); Seki, H.; Imamura, M. (Institute of Physical and Chemical Research, Wako, Saitama (Japan))


    The reaction of lysozyme with OH radical, Br/sub 2/ anion radical, and e/sup -/sub(aq), produced in an aqueous solution by pulsed electrons and ..gamma..-rays, were investigated. Irradiated enzymes showed an increase in the light scattering intensity (LSI) which is proportional to the absorbed dose. Results obtained from SDS gel electrophoresis confirm dimerization of lysozyme, which is considered to be responsible for the increase in LSI. It was found that the rate constant of the dimerization of protein radicals produced in the reaction with OH radical is 2k = (1.0+-0.3) x 10/sup 6/M/sup -1/s/sup -1/ and the yield of the dimerization is 0.6 in G. The enzymatic activity of the dimer is shown to be reduced to about 30 per cent of that of the intact enzyme. It is concluded that the radiation-induced inactivation of lysozyme is largely due to dimerization.

  1. Dimer formation in radiation-irradiated aqueous solution of lysozyme studied by light-scattering-intensity measurement. (United States)

    Hashimoto, S; Seki, H; Masuda, T; Imamura, M; Kondo, M


    The reaction of lysozyme with OH., Br.-2 and e-aq, produced in an aqueous solution by pulsed electrons and gamma-rays, were investigated. Irradiated enzymes showed an increase in the light scattering intensity (LSI) which is proportional to the absorbed dose. Results obtained from SDS gel electrophoresis confirm dimerization of lysozyme, which is considered to be responsible for the increase in LSI. It was found that the rate constant of the dimerization of protein radicals produced in the reaction with OH. is 2K=(1.0 +/- 0.3) X 10(6)M-1 s-1 and the yield of the dimerization is 0.6 in G. The enzymatic activity of the dimer is shown to be reduced to about 30 per cent of that of the intact enzyme. It is concluded that the radiation-induced inactivation of lysozyme is largely due to dimerization.

  2. The Effects of Thermal History on Nucleation of Tetragonal Lysozyme Crystals, or Hot Protein and Cold Nucleation (United States)

    Burke, Michael; Judge, Russell; Pusey, Marc


    Chicken egg white lysozyme has a well characterized thermally driven phase transition. Between pH 4.2 and 5.2, the transition temperature, as defined by the point where the tetragonal and orthorhombic solubilities are equal, is a function of the pH, salt (precipitant) type and concentration, and most likely of the buffer concentration as well. This phase transition can be carried out with protein solution alone, prior to addition of precipitant solution. Warming a lysozyme solution above the phase transition point, then cooling it back below this point, has been shown to affect the subsequent nucleation rate, as determined by the numbers and size of crystals formed, but not the growth rate for the tetragonal crystal form . We have now measured the kinetics of this process and investigated its reversibility. The transition effects are progressive with temperature, having a half time of about 1 hour at 37C at pH 4.8. After holding a lysozyme solution at 37C (prior to addition of precipitant) for 16 hours, then cooling it back to 4C no return to the pre-warmed nucleation kinetics are observed after at least 4 weeks. Orthorhombic lysozyme crystals apparently do not undergo the flow-induced growth cessation of tetragonal lysozyme crystals. Putting the protein in the orthorhombic form does not affect the averaged face growth kinetics, only nucleation, for tetragonal crystals. This differential behaviour may be exploited to elucidate how and where flow affects the lysozyme crystal growth process. The presentation will focus on the results of these and ongoing studies in this area.

  3. Expression of lysozymes from Erwinia amylovora phages and Erwinia genomes and inhibition by a bacterial protein. (United States)

    Müller, Ina; Gernold, Marina; Schneider, Bernd; Geider, Klaus


    Genes coding for lysozyme-inhibiting proteins (Ivy) were cloned from the chromosomes of the plant pathogens Erwinia amylovora and Erwinia pyrifoliae. The product interfered not only with activity of hen egg white lysozyme, but also with an enzyme from E. amylovora phage ΦEa1h. We have expressed lysozyme genes from the genomes of three Erwinia species in Escherichia coli. The lysozymes expressed from genes of the E. amylovora phages ΦEa104 and ΦEa116, Erwinia chromosomes and Arabidopsis thaliana were not affected by Ivy. The enzyme from bacteriophage ΦEa1h was fused at the N- or C-terminus to other peptides. Compared to the intact lysozyme, a His-tag reduced its lytic activity about 10-fold and larger fusion proteins abolished activity completely. Specific protease cleavage restored lysozyme activity of a GST-fusion. The bacteriophage-encoded lysozymes were more active than the enzymes from bacterial chromosomes. Viral lyz genes were inserted into a broad-host range vector, and transfer to E. amylovora inhibited cell growth. Inserted in the yeast Pichia pastoris, the ΦEa1h-lysozyme was secreted and also inhibited by Ivy. Here we describe expression of unrelated cloned 'silent' lyz genes from Erwinia chromosomes and a novel interference of bacterial Ivy proteins with a viral lysozyme.

  4. Analysis of two lysozyme genes and antimicrobial functions of their recombinant proteins in Asian seabass.

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    Gui Hong Fu

    Full Text Available Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type and goose-type (g-type lysozymes from Asian seabass (Lates calcarifer. The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu(50 and Asp(67 and a "GSTDYGIFQINS" motif. The deduced g-type lysozyme contained 187 residues and possessed a goose egg white lysozyme (GEWL domain containing three conserved catalytic residues (Glu(71, Asp(84, Asp(95 essential for catalytic activity. Real time quantitative PCR (qRT-PCR revealed that the two lysozyme genes were constitutively expressed in all the examined tissues. The c-type lysozyme was most abundant in liver, while the g-type lysozyme was predominantly expressed in intestine and weakly expressed in muscle. The c-type and g-type transcripts were up-regulated in the kidney, spleen and liver in response to a challenge with Vibrio harveyi. The up-regulation of the c-type lysozyme was much stronger than that of the g-type lysozyme in kidney and spleen. The recombinant proteins of the c-type and g-type lysozymes showed lytic activities against the bacterial pathogens Vibrio harveyi and Photobacterium damselae in a dosage-dependent manner. We identified single nucleotide polymorphisms (SNPs in the two lysozyme genes. There were significant associations of these polymorphisms with resistance to the big belly disease. These results suggest that the c- and g-type genes play an important role in resistance to bacterial pathogens in fish. The SNP markers in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to bacterial diseases.

  5. Relaxation dynamics of lysozyme in solution under pressure: Combining molecular dynamics simulations and quasielastic neutron scattering

    Energy Technology Data Exchange (ETDEWEB)

    Calandrini, V. [Centre de Biophysique Moleculaire, Rue Charles Sadron, 45071 Orleans (France); Synchrotron Soleil, L' Orme de Merisiers, B.P. 48, 91192 Gif-sur-Yvette (France); Hamon, V. [Centre de Biophysique Moleculaire, Rue Charles Sadron, 45071 Orleans (France); Hinsen, K. [Centre de Biophysique Moleculaire, Rue Charles Sadron, 45071 Orleans (France); Synchrotron Soleil, L' Orme de Merisiers, B.P. 48, 91192 Gif-sur-Yvette (France); Calligari, P. [Centre de Biophysique Moleculaire, Rue Charles Sadron, 45071 Orleans (France); Institut Laue-Langevin, 6 Rue Jules Horowitz, B.P. 156, 38042 Grenoble (France); Laboratoire Leon Brillouin, CEA Saclay, 91191 Gif-sur-Yvette (France); Bellissent-Funel, M.-C. [Laboratoire Leon Brillouin, CEA Saclay, 91191 Gif-sur-Yvette (France); Kneller, G.R. [Centre de Biophysique Moleculaire, Rue Charles Sadron, 45071 Orleans (France); Synchrotron Soleil, L' Orme de Merisiers, B.P. 48, 91192 Gif-sur-Yvette (France)], E-mail:


    This paper presents a study of the influence of non-denaturing hydrostatic pressure on the relaxation dynamics of lysozyme in solution, which combines molecular dynamics simulations and quasielastic neutron scattering experiments. We compare results obtained at ambient pressure and at 3 kbar. Experiments have been performed at pD 4.6 and at a protein concentration of 60 mg/ml. For both pressures we checked the monodispersity of the protein solution by small angle neutron scattering. To interpret the simulation results and the experimental data, we adopt the fractional Ornstein-Uhlenbeck process as a model for the internal relaxation dynamics of the protein. On the experimental side, global protein motions are accounted for by the model of free translational diffusion, neglecting the much slower rotational diffusion. We find that the protein dynamics in the observed time window from about 1 to 100 ps is slowed down under pressure, while its fractal characteristics is preserved, and that the amplitudes of the motions are reduced by about 20%. The slowing down of the relaxation is reduced with increasing q-values, where more localized motions are seen.

  6. Viscometric study of lysozyme solution with sugar and urea at various temperatures

    Directory of Open Access Journals (Sweden)

    Jamal Akhter Siddique


    at temperatures (293.15, 303.15, 313.13 and 323.15 K at various concentrations of glucose, maltose and urea. Change in entropy (ΔH, enthalpy (ΔS and free energy of activation (ΔG have also been evaluated for these systems. Value of B-coefficient of d (− glucose, maltose and urea has also been calculated from viscosity data in aqueous lysozyme solution. Viscosity B-coefficients of glucose and maltose in aqueous lysozyme solution are positive while that of the urea–lysozyme water system it is negative due to the structure breaking effect of urea. The values of entropy of activation are negative due to attainment of transition state for viscous flow, which is accompanied by bond formation and increase in order.

  7. Complex coacervates of hyaluronic acid and lysozyme

    DEFF Research Database (Denmark)

    Water, Jorrit J.; Schack, Malthe M.; Velazquez-Campoy, Adrian


    by differential scanning calorimetry. Furthermore, the protein stability of lysozyme was found to be improved upon complexation during a 12-weeks storage study at room temperature, as shown by a significant increase in recovered protein when complexed (94 ± 2% and 102 ± 5% depending on the polymer-protein weight...... stoichiometry was determined using solution depletion and isothermal titration calorimetry. The binding stoichiometry of lysozyme to hyaluronic acid (870 kDa) determined by solution depletion was found to be 225.9 ± 6.6 mol, or 0.1 bound lysozyme molecules per hyaluronic acid monomer. This corresponded well...... with that obtained by isothermal titration calorimetry of 0.09 bound lysozyme molecules per hyaluronic acid monomer. The complexation did not alter the secondary structure of lysozyme measured by Fourier-transform infrared spectroscopy overlap analysis and had no significant impact on the Tm of lysozyme determined...

  8. The antibacterial protein lysozyme identified as the termite egg recognition pheromone.

    Directory of Open Access Journals (Sweden)

    Kenji Matsuura

    Full Text Available Social insects rely heavily on pheromone communication to maintain their sociality. Egg protection is one of the most fundamental social behaviours in social insects. The recent discovery of the termite-egg mimicking fungus 'termite-ball' and subsequent studies on termite egg protection behaviour have shown that termites can be manipulated by using the termite egg recognition pheromone (TERP, which strongly evokes the egg-carrying and -grooming behaviours of workers. Despite the great scientific and economic importance, TERP has not been identified because of practical difficulties. Herein we identified the antibacterial protein lysozyme as the TERP. We isolated the target protein using ion-exchange and hydrophobic interaction chromatography, and the MALDI-TOF MS analysis showed a molecular size of 14.5 kDa. We found that the TERP provided antibacterial activity against a gram-positive bacterium. Among the currently known antimicrobial proteins, the molecular size of 14.5 kDa limits the target to lysozyme. Termite lysozymes obtained from eggs and salivary glands, and even hen egg lysozyme, showed a strong termite egg recognition activity. Besides eggs themselves, workers also supply lysozyme to eggs through frequent egg-grooming, by which egg surfaces are coated with saliva containing lysozyme. Reverse transcript PCR analysis showed that mRNA of termite lysozyme was expressed in both salivary glands and eggs. Western blot analysis confirmed that lysozyme production begins in immature eggs in queen ovaries. This is the first identification of proteinaceous pheromone in social insects. Researchers have focused almost exclusively on hydrocarbons when searching for recognition pheromones in social insects. The present finding of a proteinaceous pheromone represents a major step forward in, and result in the broadening of, the search for recognition pheromones. This novel function of lysozyme as a termite pheromone illuminates the profound influence

  9. The influence of a homologous protein impurity on lysozyme crystal growth (United States)

    Bhamidi, V.; Hanson, B. L.; Edmundson, A.; Skrzypczak-Jankun, E.; Schall, C.


    The effect of a structurally similar protein impurity, turkey ( Meleagris gallopavo) egg-white lysozyme (TEWL) on crystallization of the host protein, hen-egg-white lysozyme (HEWL) from chicken ( Gallus gallus) was studied under varying impurity and host solution concentrations. A change in morphology is observed when crystals of HEWL are grown in the presence of TEWL. As the relative amount of TEWL increases, HEWL crystals become more elongated in the [0 0 1] direction. Elongation is more pronounced in samples with lower initial concentrations of HEWL than in samples with higher initial concentrations. This behavior is consistent with that of impurities in small molecule crystal growth and with predictions based on the Kubota-Mullin model. The observed effect on the growth process can be attributed to the apparent inhibition in the [1 1 0] crystal growth direction of HEWL by TEWL since slowly growing faces become dominant faces in crystal growth. Incorporation of TEWL into HEWL crystals grown in a sitting drop batch method was measured using cation exchange chromatography. The results indicate that impurity incorporation is associated with increasing supersaturation. This conclusion is consistent with a kinetically controlled process of impurity incorporation. The observed impurity effects are most probably associated with the interchange of glutamine in position 41 of HEWL by histidine in TEWL.

  10. Lysozyme coated DNA and DNA/SWNT fibers by solution spinning. (United States)

    Nepal, Dhriti; Minus, Marilyn L; Kumar, Satish


    DNA fibers were prepared by solution spinning of DNA in a lysozyme (LSZ) coagulation/gelation bath. Strong positive charges carried by LSZ protein condensed the DNA (strong negative charged) molecules resulting in self-assembly and the formation of fibrillar structures in a gel-like network. DNA/LSZ fibril formation was found to be dependent on the ratio of DNA to LSZ. A minimum 0.1 wt.-% of LSZ was necessary to condense 0.1 wt.-% of DNA into micro-fibrils. Macroscopic fiber spinning was possible by introducing a 0.1 wt.-% DNA aqueous solution into a 0.2 wt.-% LSZ coagulation bath which resulted in fibers with ≈20 µm diameter. Single-walled carbon nanotubes (SWNT) were also incorporated into these fibers to explore the possibility for creating hybrid materials. All DNA-based fibers exhibit strong birefringence confirming molecular orientation along the fiber axis. Due to the presence of LSZ, the fibers exhibit antimicrobial activity against bacteria like Micrococcus lysodeikticus.

  11. Octamer formation in lysozyme solutions at the initial crystallization stage detected by small-angle neutron scattering. (United States)

    Boikova, Anastasiia S; Dyakova, Yulia A; Ilina, Kseniia B; Konarev, Petr V; Kryukova, Alyona E; Kuklin, Alexandr I; Marchenkova, Margarita A; Nabatov, Boris V; Blagov, Alexandr E; Pisarevsky, Yurii V; Kovalchuk, Mikhail V


    Solutions of lysozyme in heavy water were studied by small-angle neutron scattering (SANS) at concentrations of 40, 20 and 10 mg ml(-1) with and without the addition of precipitant, and at temperatures of 10, 20 and 30°C. In addition to the expected protein monomers, dimeric and octameric species were identified in solutions at the maximum concentration and close to the optimal conditions for crystallization. An optimal temperature for octamer formation was identified and both deviation from this temperature and a reduction in protein concentration led to a significant decrease in the volume fractions of octamers detected. In the absence of precipitant, only monomers and a minor fraction of dimers are present in solution.

  12. The Release of Egg White Lysozyme Containing EDTA from Composite Edible Film Based on Whey Protein, Konjac Flour and Lipid

    Directory of Open Access Journals (Sweden)

    Mulia W. Apriliyani


    Full Text Available The objectives of this research were to find out the effect of EDTA addition on antibacterial spectrum broadening of lysozyme on Gram negative bacteria and the release of lysozyme from composite edible film made of whey protein, konjac glucomannan and several lipids type and content. The research were conducted with 2 steps. Step I: The addition of EDTA on lysozyme aquaeous (Lysozyme (mg/mL: EDTA (mg/mL = 11.14:8.14; 11.14:11.14 and 11.14:14.14 using Randomyzed Block Design, the variables were, antibacterial of lysozyme on Micrococcus lysodeikticus and Escherichia coli. Step II: Lipid content (5 and 10% and kind of lipid (butter, margarine, palm oil and beeswax using nested Randomyzed Block Design, the variables were lysozyme release, Water Vapor Permeability (WVP, protein solublity and microstructure of composite edible film. The results were, step I: the treatment didn’t gave significantly effect (p>0.05 on lysozyme activity. EDTA decrease cell membrane stabilization and lysozyme made lysis of cell membrane. EDTA chelate Ca2+ and Mg2+ salts as bridge between Lypopolysachcharide (LPS in outer membrane so LPS released from cell wall of Gram negative bacteria. Step II: The treatment didn’t gave significantly effect (p>0.05 on release of lysozyme and water vapour permeability, but gave significantly effect (p<0.05 on protein solubility. The release of lysozyme from composite edible film gave the best lysozyme release from beeswax 10% addition.

  13. Characterizing protein activities on the lysozyme and nanodiamond complex prepared for bio applications. (United States)

    Perevedentseva, E; Cai, P-J; Chiu, Y-C; Cheng, C-L


    Recently, nanodiamond particles have attracted increasing attention as a promising nanomaterial for its biocompatibility, easy functionalization and conjugation with biomolecules, and its superb physical/chemical properties. Nanodiamonds are mainly used as markers for cell imaging, using its fluorescence or Raman signals for detection, and as carriers for drug delivery. For the success of these applications, the biomolecule associated with the nanodiamond has to retain its functionality. In this work, the protein activities of egg white lysozyme adsorbed on nanodiamond particles of different sizes is investigated. The lysozyme nanodiamond complex is used here as a protein model for analyzing its structural conformation changes and, correspondingly, its enzymatic activity after the adsorption. Fourier-transform infrared spectroscopy (FTIR) is used for the analysis of the sensitive protein secondary structure. To access the activities of the adsorbed lysozyme, a fluorescence-based assay is used. The process of adsorption is also analyzed using UV-visible spectroscopic measurements in combination with analysis of nanodiamond properties with FTIR, Raman spectroscopy, and ζ-potential measurements. It is found that the activity of lysozyme upon adsorption depends on the nanodiamond's size and surface properties, and that the nanodiamond particles can be selected and treated, which do not alter the lysozyme functional properties. Such nanodiamonds can be considered convenient nanoparticles for various bioapplications.

  14. High performance aptamer affinity chromatography for single-step selective extraction and screening of basic protein lysozyme. (United States)

    Han, Bin; Zhao, Chao; Yin, Junfa; Wang, Hailin


    A DNA aptamer based high-performance affinity chromatography is developed for selective extraction and screening of a basic protein lysozyme. First, a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic column was synthesized in situ by thermally initiated radical polymerization, and then an anti-lysozyme DNA aptamer was covalently immobilized on the surface of the monolith through a 16-atom spacer arm. The target protein lysozyme but non-target proteins can be trapped by the immobilized anti-lysozyme DNA aptamer. In contrast, lysozyme cannot be trapped by the immobilized oligodeoxynucleotide that does not contain the sequence of the anti-lysozyme DNA aptamer. The study clearly demonstrates the trapping of lysozyme by the immobilized anti-lysozyme DNA aptamer is mainly due to specific recognition rather than simple electrostatic interaction of positively charged protein and the negatively charged DNA. The inter-day precision was determined as 0.8% for migration time and 4.2% for peak area, respectively. By the use of aptamer affinity monolith, a screening strategy is developed to selectively extract lysozyme from chicken egg white, showing the advantages of high efficiency, low cost and ease-of-operation.

  15. Release of lysozyme from electrospun PVA/lysozyme-gelatin scaffolds

    Institute of Scientific and Technical Information of China (English)

    Dong-zhi YANG; Yu-hua LONG; Jun NIE


    This article describes an electrospinning process in fabricating ultra fine fibers with core-shell structure. A biodegradable polymer, poly(vinyl alcohol) (PVA), was used as the shell; lysozyme was a kind of antioxidant; and gelatin were used as the core. Morphology and microstruc-ture of the ultra fine fibers were characterized by scanning electron microscope (SEM), transmission electron micro-scopy (TEM) and X-ray photoelectron spectroscopy (XPS) analysis. As a comparison, composite nanofiber PVA/lysozyme-gelatin blend was prepared by a normal electrospinning process. In vitro drug release behaviors of the nanofibrous membranes were determined in phosphate-buffered saline (PBS) solution. It was found that core-shell nanofibers PVA/lysozyme-gelatin obviously exhibit higher initial release rates compared to that of PVA/lysozyme-gelatin blend nanofibers. The current method may find wide application in controlled release of bioactive proteins and tissue engineering.

  16. Identification of salivary proteins at oil–water interfaces stabilized by lysozyme and ß-lactoglobulin

    NARCIS (Netherlands)

    Silletti, E.; Vitorino, R.M.P.; Schipper, R.G.; Amado, F.M.L.; Vingerhoeds, M.H.


    In this research, we investigated the interaction occurring between oil-in-water emulsion droplets, stabilized by different emulsifiers, i.e. lysozyme and ß-lactoglobulin (ß-lg), and salivary proteins (SPs) with a molecular mass (Mr) above about 10 kDa. Different techniques, i.e. infrared

  17. Thin films of protein (BSA, lysozyme) - Polyelectrolyte (PSS) complexes show larger red-shift in optical emissions irrespective of protein conformation (United States)

    Talukdar, Hrishikesh; Kundu, Sarathi


    Protein-polyelectrolyte complexes (PPC) are prepared using globular proteins (BSA, lysozyme) and optically active polyelectrolyte poly (sodium 4-styrenesulfonate) (PSS) in aqueous solutions and as thin films on solid substrates to explore their structures and optical behaviors. Out-of-plane structures of PPC films having ≈15-60 nm thicknesses are investigated from X-ray reflectivity and their relatively smooth surface morphologies are obtained from atomic force microscopy. ATR-FTIR spectroscopy confirms that the conformation of BSA proteins inside the films of PSSB (PSS+BSA) is nearly same with pure BSA but for lysozyme inside PSSL (PSS+lysozyme) conformation modifies which is evidenced from the shifting of the amide-I band of each protein. However, irrespective of the conformation variation of proteins larger red-shifts of ≈30 nm in optical emissions are obtained from the thin films of PPC. Relatively enhance dissipation of energy thorough non-radiative transition of the fluorophore residues in the dry state is the most probable reason for such larger optical red-shifts.

  18. The Effect of Solution Parameters on Lysozyme Nucleation Rates and Crystal Quality (United States)

    Judge, R. A.; Snell, E. H.


    In the pursuit of strongly diffracting high quality macromolecule crystals of suitable volume, this study investigates how the formation of macromolecules in solution and their growth characteristics effect crystal volume and diffracting quality. We systematically investigated the effect of solution conditions on lysozyme nucleation rates and the volume of crystals produced. Batch crystallization plates were used in combination with a video microscope system to measure nucleation rates and crystal volume. As expected from classical nucleation theory, crystal numbers were found to increase with increases in temperature and supersaturation. Small changes in solution pH, at constant supersaturation values were found, however, to dramatically effect the number of crystals nucleated in the wells varying from 1000s to 10s in the pH range 4.0 to 5.2. Having optimized the conditions required to produce an appropriate number of crystals of a suitable volume for X-ray analysis, a large number of uniform crystals were produced under exactly the same conditions. In the X-ray analysis of more than 50 such crystals there was found a wide variation in crystal lattice parameters and data quality. The variation in X-ray quality crystal samples is thought to be related to the growth rate variation caused by growth rate dispersion seen in lysozyme crystal growth experiments.

  19. Size-dependent interaction of silica nanoparticles with lysozyme and bovine serum albumin proteins (United States)

    Yadav, Indresh; Aswal, Vinod K.; Kohlbrecher, Joachim


    The interaction of three different sized (diameter 10, 18, and 28 nm) anionic silica nanoparticles with two model proteins—cationic lysozyme [molecular weight (MW) 14.7 kDa)] and anionic bovine serum albumin (BSA) (MW 66.4 kDa) has been studied by UV-vis spectroscopy, dynamic light scattering (DLS), and small-angle neutron scattering (SANS). The adsorption behavior of proteins on the nanoparticles, measured by UV-vis spectroscopy, is found to be very different for lysozyme and BSA. Lysozyme adsorbs strongly on the nanoparticles and shows exponential behavior as a function of lysozyme concentration irrespective of the nanoparticle size. The total amount of adsorbed lysozyme, as governed by the surface-to-volume ratio, increases on lowering the size of the nanoparticles for a fixed volume fraction of the nanoparticles. On the other hand, BSA does not show any adsorption for all the different sizes of the nanoparticles. Despite having different interactions, both proteins induce similar phase behavior where the nanoparticle-protein system transforms from one phase (clear) to two phase (turbid) as a function of protein concentration. The phase behavior is modified towards the lower concentrations for both proteins with increasing the nanoparticle size. DLS suggests that the phase behavior arises as a result of the nanoparticles' aggregation on the addition of proteins. The size-dependent modifications in the interaction potential, responsible for the phase behavior, have been determined by SANS data as modeled using the two-Yukawa potential accounting for the repulsive and attractive interactions in the systems. The protein-induced interaction between the nanoparticles is found to be short-range attraction for lysozyme and long-range attraction for BSA. The magnitude of attractive interaction irrespective of protein type is enhanced with increase in the size of the nanoparticles. The total (attractive+repulsive) potential leading to two-phase formation is found to be

  20. Pulsed laser deposition of the lysozyme protein: an unexpected “Inverse MAPLE” process

    DEFF Research Database (Denmark)

    Schou, Jørgen; Matei, Andreea; Constantinescu, Catalin


    the ejection and deposition of lysozyme. This can be called an “inverse MAPLE” process, since the ratio of “matrix” to film material in the target is 10:90, which is inverse of the typical MAPLE process where the film material is dissolved in the matrix down to several wt.%. Lysozyme is a well-known protein...... which is used in food processing and is also an important constituent of human secretions such as sweat and saliva. It has a well-defined mass (14307 u) and can easily be detected by mass spectrometric methods such as MALDI (Matrix-assisted laser desorption ionization) in contrast to many other organic...... materials. Also, the thermal properties of lysozyme, including the heat-induced decomposition behavior are comparatively well-known. The ablation of lysozyme from a dry pressed target in vacuum was measured by weight loss in nanosecond laser ablation at 355 with a fluence of 0.5 to 6 J/cm2. Films...

  1. Effects of protein and phosphate buffer concentrations on thermal denaturation of lysozyme analyzed by isoconversional method. (United States)

    Cao, X M; Tian, Y; Wang, Z Y; Liu, Y W; Wang, C X


    Thermal denaturation of lysozymes was studied as a function of protein concentration, phosphate buffer concentration, and scan rate using differential scanning calorimetry (DSC), which was then analyzed by the isoconversional method. The results showed that lysozyme thermal denaturation was only slightly affected by the protein concentration and scan rate. When the protein concentration and scan rate increased, the denaturation temperature (Tm) also increased accordingly. On the contrary, the Tm decreased with the increase of phosphate buffer concentration. The denaturation process of lysozymes was accelatated and the thermal stability was reduced with the increase of phosphate concentration. One part of degeneration process was not reversible where the aggregation occurred. The other part was reversible. The apparent activation energy (Ea) was computed by the isoconversional method. It decreased with the increase of the conversion ratio (α). The observed denaturation process could not be described by a simple reaction mechanism. It was not a process involving 2 standard reversible states, but a multi-step process. The new opportunities for investigating the kinetics process of protein denaturation can be supplied by this novel isoconversional method.

  2. T4 phage lysozyme: a protein designed for understanding tryptophan photophysics (United States)

    Hudson, Bruce S.; Harris, Dan


    Bacteriophage T4 lysozyme in its wild type form contains three tryptophan residues (at sequence postions 126, 138 and 158). These three residues are in rather different environments in the protein: 126 and 158 are near the protein surface while residue 138 is more buried. T4 lysozyme has been genetically engineered to prepare all possible variants in which one or more of the tryptophan residues have been replaced by tyrosine. The available data supports the hypothesis that this substitution has, at most, a very minor effect on the structure of the protein. The three species with single tryptophan residues have been investigated in detail. The surface location of residue 126 compared to the buried location of residue 138 is reflected in the difference in collisional quenching observed with added potassium iodide. It is found that the spectral and radiative properties of the three proteins are very similar but that their radiationless decay properties are quite distinct. This is apparently due to short-range collisional quenching by neighboring side chains. Comparison with solution quenching measurements permits the identification of the specific quenching groups involved for each tryptophan residue and provides a semi-quantitative rationale for the radiationless decay rate. This collisional quenching interpretation is supported by mutational effects on fluorescence quantum yield. This simple picture of the behavior of these single-tryptophan proteins is clearly revealed in this particular case because of the unambiguous choice of collisional quenching groups. The time dependence of the fluorescence decay of each of these single-tryptophan proteins is quite complex. Several methods of analysis are presented and discussed in terms of their underlying physical basis. Internal collisional quenching, as suggested from the comparative studies, is expected to lead to non-exponential behavior. This is consistent with the observed time dependence. Analysis of the temporal

  3. Structural, Functional and Phylogenetic Analysis of Sperm Lysozyme-Like Proteins. (United States)

    Kalra, Shalini; Pradeep, Mangottil Ayyappan; Mohanty, Ashok K; Kaushik, Jai K


    Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6 are expressed in the testis of mammals. Not all members of LYZL family have been uniformly and unambiguously identified in the genome and proteome of mammals. Some studies suggested a role of SLLP1 and LYZL4 in fertilization; however, the function of other LYZL proteins is unknown. We identified all known forms of LYZL proteins in buffalo sperm by LC-MS/MS. Cloning and sequence analysis of the Lyzl cDNA showed 38-50% identity at amino acid level among the buffalo LYZL paralogs, complete conservation of eight cysteines and other signature sequences of c-type lysozyme family. Catalytic residues in SLLP1, LYZL4 and LYZL5 have undergone replacement. The substrate binding residues showed significant variation in LYZL proteins. Residues at sites 62, 101, 114 in LYZL4; 101 in SLLP1; 37, 62, and 101 in LYZL6 were more variable among diverse species. Sites 63 and 108 occupied by tryptophan were least tolerant to variation. Site 37 also showed lower tolerance to substitution in SLLP1, LYZL4 and LYZL5, but more variable in non-testicular lysozymes. Models of LYZL proteins were created by homology modeling and the substrate binding pockets were analyzed in term of binding energies and contacting residues of LYZL proteins with tri-N-acetylglucosamine (NAG)3 in the A-B-C and B-C-D binding mode. Except LYZL6, LYZL proteins did not show significant difference in binding energies in comparison to hen egg white lysozyme in the A-B-C mode. (NAG)3 binding energy in the B-C-D mode was higher by 1.3-2.2 kcal/mol than in A-B-C mode. Structural analysis indicated that (NAG)3 was involved in making more extensive interactions including hydrogen bonding with LYZL proteins in B-C-D mode than in A-B-C mode. Despite large sequence divergence among themselves and with respect to c-type lysozymes, substrate binding residues as

  4. N-terminal T4 lysozyme fusion facilitates crystallization of a G protein coupled receptor.

    Directory of Open Access Journals (Sweden)

    Yaozhong Zou

    Full Text Available A highly crystallizable T4 lysozyme (T4L was fused to the N-terminus of the β(2 adrenergic receptor (β(2AR, a G-protein coupled receptor (GPCR for catecholamines. We demonstrate that the N-terminal fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.

  5. New Equation of State for Osmotic Pressures in Aqueous Saline Solutions of Lysozyme

    Institute of Scientific and Technical Information of China (English)

    林阳政; 李以圭; 陆九芳


    A new equation of state is proposed to correlate the osmotic pressure data for aqueous lysozyme solutions with (NH4)2SO4, (NH4)2C2O4 and (NH4)2HPO4 at ionic strengths of 13.5 mol/kg and pH 4, 7 or 8 with only one adjustable parameter instead of the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The Carnahan-Starling equation represents the contribution of the hard sphere repulsion to the osmotic pressure. The attractive dispersion interaction is represented by the Lennard-Jones potential expressed by the equation of Cotterman et al. based on perturbation theory. The double-layer repulsion interaction is represented by Yukawa potential expressed by the equation of state of Duh and Mier-Y-Teran based on mean spherical approximation. The total average relative deviation of the correlation of the osmotic pressure data is 1.68%.

  6. Effects of mannose, fructose, and fucose on the structure, stability, and hydration of lysozyme in aqueous solution

    DEFF Research Database (Denmark)

    Rahim, Abdoul; Peters, Günther H.J.; Jalkanen, Karl J.


    The bio-protective properties of monosaccharaides, namely mannose, fructose and fucose, on the stability and dynamical properties of the NMR determined hen egg-white lysozyme structure have been investigated by means of molecular dynamics simulations at room temperature in aqueous solution and in...

  7. The interaction of lysozyme with caffeine, theophylline and theobromine in solution. (United States)

    Zhang, Hong-Mei; Tang, Bo-Ping; Wang, Yan-Qing


    The interactions of lysozyme with caffeine (Caf), theophylline (Tph) and theobromine (Tbr) were investigated using UV-Vis absorption, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques. The results revealed that Caf (Tph or Tbr) caused the fluorescence quenching of lysozyme by the formation of Caf (Tph or Tbr)-lysozyme complex. The binding constants (K(A)) and thermodynamic parameters (ΔG°, ΔH°, ΔS°) at two different temperatures, the binding locality, and the binding power were obtained. The results showed that the process of binding Caf (Tph or Tbr) to lysozyme was a spontaneous molecular interaction procedure and the hydrophobic and electrostatic interactions play a major role in stabilizing the complex; The distance r between donor (lysozyme) and acceptor (Caf, Tph or Tbr) was obtained according to fluorescence resonance energy transfer. The effect of Caf (Tph or Tbr) on the conformation of lysozyme was analyzed using synchronous fluorescence and three-dimensional fluorescence spectra techniques. The results showed that the binding of Caf (Tph or Tbr) to lysozyme induced some micro-environmental and conformational changes in lysozyme and disturbed the environment of the polypeptide of lysozyme.

  8. Pulsed laser deposition of the lysozyme protein: an unexpected “Inverse MAPLE” process

    DEFF Research Database (Denmark)

    Schou, Jørgen; Matei, Andreea; Constantinescu, Catalin


    the ejection and deposition of lysozyme. This can be called an “inverse MAPLE” process, since the ratio of “matrix” to film material in the target is 10:90, which is inverse of the typical MAPLE process where the film material is dissolved in the matrix down to several wt.%. Lysozyme is a well-known protein...... which is used in food processing and is also an important constituent of human secretions such as sweat and saliva. It has a well-defined mass (14307 u) and can easily be detected by mass spectrometric methods such as MALDI (Matrix-assisted laser desorption ionization) in contrast to many other organic...

  9. Laser ablation of the lysozyme protein: a model system for soft materials

    DEFF Research Database (Denmark)

    Schou, Jørgen; Matei, Andreea; Constantinescu, Catalin

    ionization) in contrast to many other organic materials. Also the thermal properties, including the heat-induced decomposition behavior are comparatively well-known. For laser-irradiation at wavelengths above 310 nm, no photochemical processes occur initially, but the material is ejected via photothermal......Lysozyme is a well-known protein which is used in food processing and is also an important constituent of human secretions such as sweat and saliva. It has a well-defined mass (14307 u) and can easily be detected by mass spectrometric methods such as MALDI (Matrix-assisted laser desorption...... on the results of molecular-level modeling. In particular, the effect of the possible presence of trapped water pockets in the lysozyme targets is investigated in the simulations and the minimum amount of water required for the lift off of the intact molecules is established....

  10. Rodlike Complexes of a Polyelectrolyte (Hyaluronan) and a Protein (Lysozyme) observed by SANS

    CERN Document Server

    Boué, François; Cousin, Fabrice; Grillo, Isabelle; Morfin, Isabelle; 10.1021/bm100861g


    We study by Small Angle Neutron Scattering (SANS) the structure of Hyaluronan -Lysozyme complexes. Hyaluronan (HA) is a polysaccharide of 9 nm intrinsic persistence length that bears one negative charge per disaccharide monomer (Mmol = 401.3 g/mol); two molecular weights, Mw = 6000 and 500 000 Da were used. The pH was adjusted at 4.7 and 7.4 so that lysozyme has a global charge of +10 and + 8 respectively. The lysozyme concentration was varied from 3 to 40 g/L, at constant HA concentration (10 g/L). At low protein concentration, samples are monophasic and SANS experiments reveal only fluctuations of concentration although, at high protein concentration, clusters are observed by SANS in the dense phase of the diphasic samples. In between, close to the onset of the phase separation, a distinct original scattering is observed. It is characteristic of a rod-like shape, which could characterize "single" complexes involving one or a few polymer chains. For the large molecular weight (500 000) the rodlike rigid doma...

  11. Evaluation of lysozyme, complement C3, and total protein in different developmental stages of Caspian kutum (Rutilus frisii kutum K.

    Directory of Open Access Journals (Sweden)

    Abdollahi Razieh


    Full Text Available In this study, non–specific immune parameters in fertilized eggs, eyed embryos, larvae 10, 25, 50, 60, and 70 days post hatch (DPH, and female broodstock of Caspian kutum, Rutilus frisii kutum (Kamensky, were evaluated. The lysozyme activity, complement C3, and total protein levels were measured with the turbidimetric, immunoturbidimetric, and Bradford methods, respectively. The results showed that lysozyme levels decreased from levels noted in the fertilized eggs until the larvae were 10 days old. Subsequently, significant increases in lysozyme levels were observed until 70 DPH. An increasing trend of complement component C3 was noted from the levels in fertilized eggs to 10 DPH, following which it decreased significantly. Total protein levels differed significantly in early developmental stages of Caspian kutum. The higher values of complement component C3 than of lysozyme in the early life stages could be indicative of the former’s more fundamental role.

  12. Solvent interactions and protein dynamics in spin-labeled T4 lysozyme. (United States)

    Stoica, I


    Aspects of T4 lysozyme dynamics and solvent interaction are investigated using atomically detailed Molecular Dynamics (MD) simulations. Two spin-labeled mutants of T4 lysozyme are analyzed (T4L-N40C and T4L-K48C), which have been found from electronic paramagnetic resonance (EPR) experiments to exhibit different mobilities at the site of spin probe attachment (N- and C-terminus of helix B, respectively). Similarities and differences in solvent distribution and diffusion around the spin label, as well as around exposed and buried residues within the protein, are discussed. The purpose is to capture possible strong interactions between the spin label (ring) and solvent molecules, which may affect EPR lineshapes. The effect of backbone motions on the water density profiles is also investigated. The focus is on the domain closure associated with the T4 lysozyme hinge-bending motion, which is analyzed by Essential Dynamics (ED). The N-terminus of helix B is found to be a "hinge" residue, which explains the high degree of flexibility and motional freedom at this site.

  13. Identifying monomer phases and cluster phases in lysozyme solutions by studying the temperature dependence of the short-time dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Baglioni, P [University of Florence; Chen, Wei-Ren [ORNL; Falus, Peter [ORNL; Faraone, Antonio [National Institute of Standards and Technology (NIST); Fratini, Emiliano [University of Florence; Hong, Kunlun [ORNL; Liu, Yun [National Institute of Standards and Technology (NIST); Porcar, L. [National Institute of Standards and Technology (NIST)


    Recently experiments that combine both small angle neutron scattering (SANS) and Neutron Spin Echo (NSE) have demonstrated that dynamic clusters can form in concentrated lysozyme solutions when there is a right combination of a short-ranged attraction and a long-ranged electrostatic repulsion. In this paper, we study the temperature effect on the dynamic cluster formation and try to pinpoint the transition concentration from a monomer phase to a cluster phase. Interestingly at even a relatively high concentration (10 % mass fraction), despite the significant change of the SANS patterns that are associated with the change of the short-ranged attraction among proteins, the normalized short-time self-diffusion coefficient is not affected. This is interpreted due to the fact that there is no cluster formation at this condition. However, at larger concentrations such as 17.5 % and 22.5 % mass fraction, we show that the average hydrodynamic radius increase significantly and causes a large decrease of the normalized self-diffusion coefficient when the temperature is changed from 25 oC to 5 oC indicating the formation of dynamic clusters in solution.

  14. Synthesis Optimisation of Lysozyme Monolayer-Coated Silver Nanoparticles in Aqueous Solution

    Directory of Open Access Journals (Sweden)

    A. V. Yakovlev


    Full Text Available This paper presents an optimisation of the synthesis of silver nanoparticles encapsulated in a biological shell. The synthesis was carried out in an aqueous solution of silver nitrate. Sodium borohydride was used as a reducing agent. Lysozyme served as a bioactive coating agent. The samples produced were studied using dynamic light scattering, transmission electron microscopy, and UV-Vis spectroscopy. The function of the dependence of the reagent ratio in obtained sols on optical properties is shown. Furthermore, the influence of the synthesis temperature, reactant ratio, and order of mixing on the particle size distribution parameters is shown. The optimal reagent mass ratio, NaBH4 : LYZ : AgNO3 = 0.22 : 0.77 : 1, is established. The resulting composition allows the synthesis of particles with a mean diameter of 18 nm and a bioshell thickness of ≈3.5 nm. Moreover, the necessity of the synthesis optimisation and precise parameter control is clearly demonstrated.

  15. Protein adsorption on a hydrophobic surface: a molecular dynamics study of lysozyme on graphite. (United States)

    Raffaini, Giuseppina; Ganazzoli, Fabio


    Adsorption of human lysozyme on hydrophobic graphite is investigated through atomistic computer simulations with molecular mechanics (MM) and molecular dynamics (MD) techniques. The chosen strategy follows a simulation protocol proposed by the authors to model the initial and the final adsorption stage on a bare surface. Adopting an implicit solvent and considering 10 starting molecular orientations so that all the main sides of the protein can face the surface, we first carry out an energy minimization to investigate the initial adsorption stage, and then long MD runs of selected arrangements to follow the surface spreading of the protein maximizing its adsorption strength. The results are discussed in terms of the kinetics of surface spreading, the interaction energy, and the molecular size, considering both the footprint and the final thickness of the adsorbed protein. The structural implications of the final adsorption geometry for surface aggregation and nanoscale structural organization are also pointed out. Further MD runs are carried out in explicit water for the native structure and the most stable adsorption state to assess the local stability of the geometry obtained in implicit solvent, and to calculate the statistical distribution of the water molecules around the whole lysozyme and its backbone.

  16. Larger red-shift in optical emissions obtained from the thin films of globular proteins (BSA, lysozyme) - polyelectrolyte (PAA) complexes (United States)

    Talukdar, Hrishikesh; Kundu, Sarathi; Basu, Saibal


    Globular proteins (lysozyme and BSA) and polyelectrolyte (sodium polyacrylic acid) are used to form protein-polyelectrolyte complexes (PPC). Out-of-plane structures of ≈30-60 nm thick PPC films and their surface morphologies have been studied by using X-ray reflectivity and atomic force microscopy, whereas optical behaviors of PPC and protein conformations have been studied by using UV-vis, photoluminescence and FTIR spectroscopy respectively. Our study reveals that thin films of PPC show a larger red-shift of 23 and 16 nm in the optical emissions in comparison to that of pure protein whereas bulk PPC show a small blue-shift of ≈3 nm. A small amount of peak-shift is found to occur due to the heat treatment or concentration variation of the polyelectrolyte/protein in bulk solution but cannot produce such film thickness independent larger red-shift. Position of the emission peak remains nearly unchanged with the film thickness. Mechanism for such larger red-shift has been proposed.

  17. Data supporting the effects of lysozyme on mRNA and protein expression in a colonic epithelial scratch wound model

    Directory of Open Access Journals (Sweden)

    Sarah K. Abey


    Full Text Available Colonic epithelial health is implicated in a host of gastrointestinal (GI diseases and disorders. Lysozyme is suspected to play a role in the ability of the epithelium to recover from injury (Abey et al., in press; Gallo, 2012; Rubio, 2014 [1–3]. Disrupted repair mechanisms may lead to delayed or ineffective recovery and disruptions to epithelial biology resulting in GI symptoms and altered barrier function (Peterson and Artis, 2014 [4]. The effect of lysozyme on the transcriptomic and proteomic profile of healthy colonic epithelial cells was investigated. Epithelial cells in culture were scratch wounded and treated with lysozyme. mRNA and protein profiles were simultaneously quantified in the same sample using a digital counting technology. Gene and protein expressions altered by the presence or absence of lysozyme are described in this article. Extensive statistical and bioinformatic analysis, and interpretation of the results can be found in “Lysozyme association with circulating RNA, extracellular vesicles, and chronic stress” (Abey et al., in press [1].

  18. Variations of serum and mucus lysozyme activity and total protein content in the male and female Caspian kutum (Rutilus frisii kutum, Kamensky 1901) during reproductive period. (United States)

    Ghafoori, Zomorod; Heidari, Behrooz; Farzadfar, Fariba; Aghamaali, Mahmoudreza


    Serum and mucus lysozyme were measured in male and female Caspian kutum (Rutilus frisii kutum) under seasonal temperature, gonadal growth and reproductive migration. Significant difference with almost similar trend in serum and mucus lysozyme of the female Caspian kutum in sampling time and ovarian growth was observed. However, while there was no significant difference in serum lysozyme of the male specimen in sampling time and testicular growth, significant variations was observed in mucus lysozyme. In addition, there was significant difference in mucus total protein both for male and female specimens. The effectiveness ratio of factors on lysozyme variations followed in descending order by seasonal temperature (main factor), reproductive activity and migration with negligible effect and the lysozyme level was not significantly different in male and female Caspian kutum.

  19. The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

    Directory of Open Access Journals (Sweden)

    Qingwen Jin

    Full Text Available Insertion of T4 lysozyme (T4L into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed.We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects.Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1 infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5.Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

  20. Correlation of Conformational Changes and Protein Degradation with Loss of Lysozyme Activity Due to Chlorine Dioxide Treatment. (United States)

    Ooi, Beng Guat; Branning, Sharon Alyssa


    Chlorine dioxide (ClO2) is a potent oxidizing agent used for the treatment of drinking water and decontamination of facilities and equipment. The purpose of this research is to elucidate the manner in which ClO2 destroys proteins by studying the effects of ClO2 on lysozyme. The degree of enzyme activity lost can be correlated to the treatment time and levels of the ClO2 used. Lysozyme activity was drastically reduced to 45.3% of original enzyme activity when exposed to 4.3 mM ClO2 in the sample after 3 h. Almost all activities were lost in 3 h after exposure to higher ClO2 concentrations of up to 16.8 and 21.9 mM. Changes in protein conformation and amount as a result of ClO2 treatment were determined using the Raman spectroscopy and gel electrophoresis. Raman shifts and the alteration of spectral features observed in the ClO2-treated lysozyme samples are associated with loss of the α-helix secondary structure, tertiary structure, and disulfide bond. Progressive degradation of the denatured lysozyme by increasing levels of chlorine dioxide was also observed in gel electrophoresis. Hence, ClO2 can effectively cause protein denaturation and degradation resulting in loss of enzyme activity.

  1. Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form. (United States)

    Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W


    Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.

  2. Effects of single-walled carbon nanotubes on lysozyme gelation. (United States)

    Tardani, Franco; La Mesa, Camillo


    The possibility to disperse carbon nanotubes in biocompatible matrices has got substantial interest from the scientific community. Along this research line, the inclusion of single walled carbon nanotubes in lysozyme-based hydrogels was investigated. Experiments were performed at different nanotube/lysozyme weight ratios. Carbon nanotubes were dispersed in protein solutions, in conditions suitable for thermal gelation. The state of the dispersions was determined before and after thermal treatment. Rheology, dynamic light scattering and different microscopies investigated the effect that carbon nanotubes exert on gelation. The gelation kinetics and changes in gelation temperature were determined. The effect of carbon and lysozyme content on the gel properties was, therefore, determined. At fixed lysozyme content, moderate amounts of carbon nanotubes do not disturb the properties of hydrogel composites. At moderately high volume fractions in carbon nanotubes, the gels become continuous in both lysozyme and nanotubes. This is because percolating networks are presumably formed. Support to the above statements comes by rheology.

  3. Impact of Microscale and Pilot-Scale Freeze-Drying on Protein Secondary Structures: Sucrose Formulations of Lysozyme and Catalase. (United States)

    Peters, Björn-Hendrik; Leskinen, Jari T T; Molnár, Ferdinand; Ketolainen, Jarkko


    Microscale (MS) freeze-drying offers rapid process cycles for early-stage formulation development. The effects of the MS approach on the secondary structures of two model proteins, lysozyme and catalase, were compared with pilot-scale (PS) vial freeze-drying. The secondary structures were assessed by attenuated total reflection Fourier transformed infrared spectroscopy. Formulations were made with increasing sucrose-protein ratios. Freeze-drying protocols involved regular cooling without thermal treatment and annealing with MS and PS equipment, and cooling rate variations with the MS. Principal component analysis of smoothed second-derivative amide I spectra revealed sucrose-protein ratio-dependent shifts toward α-helical structures. Transferability of sucrose-protein formulations from MS to PS vial freeze-drying was evidenced at regular cooling rates. Local differences in protein secondary structures between the bottom and top of sucrose-catalase samples could be detected at the sucrose-catalase ratios of 1 and 2, this being related to the initial filling height and ice crystal morphology. Annealing revealed temperature, protein, formulation, and sample location-dependent effects influencing surface morphology at the top, or causing protein secondary structure perturbation at the bottom. With the MS approach, protein secondary structure differences at different cooling rates could be detected for sucrose-lysozyme samples at the sucrose-lysozyme ratio of 1.

  4. Protein dynamics by neutron scattering: The protein dynamical transition and the fragile-to-strong dynamical crossover in hydrated lysozyme

    Energy Technology Data Exchange (ETDEWEB)

    Magazù, Salvatore; Migliardo, Federica [Dipartimento di Fisica, Università di Messina, C. da Papardo n° 31, P.O. Box 55, Vill. S. Agata, 98166 Messina (Italy); Benedetto, Antonio, E-mail: [School of Physics, University College Dublin-UCD, Belfield Campus, Dublin 2 (Ireland); School of Medical Sciences, Sydney Medical School, The University of Sydney, Anderson Stuart Building F13, Sydney, NSW 2006 (Australia); Vertessy, Beata [Institute of Enzymology, Hungarian Academy of Sciences, Karolina 29, H-1113 Budapest (Hungary)


    Highlights: • The role played by the instrumental energy resolution in neutron scattering is presented. • The effect of natural bioprotectants on protein dynamics is shown. • A connection between the protein dynamical transition and the fragile-to-strong dynamical crossover is formulated. - Abstract: In this work Elastic Incoherent Neutron Scattering (EINS) results on lysozyme water mixtures in absence and in presence of bioprotectant systems are presented. The EINS data have been collected by using the IN13 and the IN10 spectrometers at the Institut Laue-Langevin (ILL, Grenoble, France) allowing to evaluate the temperature behaviour of the mean square displacement and of the relaxation time for the investigated systems. The obtained experimental findings together with theoretical calculations allow to put into evidence the role played by the spectrometer resolution and to clarify the connexion between the registered protein dynamical transition, the system relaxation time, and the instrumental energy resolution.

  5. Crystallization and evaluation of hen egg-white lysozyme crystals for protein pH titration in the crystalline state. (United States)

    Iwai, Wakari; Yagi, Daichi; Ishikawa, Takuya; Ohnishi, Yuki; Tanaka, Ichiro; Niimura, Nobuo


    To observe the ionized status of the amino acid residues in proteins at different pH (protein pH titration in the crystalline state) by neutron diffraction, hen egg-white lysozyme was crystallized over a wide pH range (2.5-8.0). Crystallization phase diagrams at pH 2.5, 6.0 and 7.5 were determined. At pH diagram, and at pH > 4.5 the border shifted to the right (higher precipitant concentration). The qualities of these crystals were characterized using the Wilson plot method. The qualities of all crystals at different pH were more or less equivalent (B-factor values within 25-40). It is expected that neutron diffraction analysis of these crystals of different pH provides equivalent data in quality for discussions of protein pH titration in the crystalline state of hen egg-white lysozyme.

  6. Solid lipid particles for oral delivery of peptide and protein drugs I - Elucidating the release mechanism of lysozyme during lipolysis

    DEFF Research Database (Denmark)

    Christophersen, Philip Carsten B; Zhang, L.; Yang, M


    The mechanism of protein release from solid lipid particles was investigated by a new lipolysis model in a biorelevant medium containing both bile salts and phospholipids. Lysozyme, a model protein, was formulated into solid lipid particles using four different types of lipids, two triglycerides...... with different chain-length of fatty acyl groups i.e. trimyristin (TG14) and tristearin (TG18), and two lipid blends dominated by diglycerides and monoglycerides, respectively. The release of lysozyme from the solid lipid particles and the lipid hydrolysis process were assessed in the lipolysis model, while...... the drug release mechanism from solid lipid particles and can potentially be used in rational selection of lipid excipients for oral delivery of peptide/protein drugs....

  7. Enterococcus faecalis zinc-responsive proteins mediate bacterial defence against zinc overload, lysozyme and oxidative stress. (United States)

    Abrantes, Marta C; Kok, Jan; Silva Lopes, Maria de Fátima


    Two Enterococcus faecalis genes encoding the P-type ATPase EF1400 and the putative SapB protein EF0759 were previously shown to be strongly upregulated in the presence of high concentrations of zinc. In the present work, we showed that a Zn(2+)-responsive DNA-binding motif (zim) is present in the promoter regions of these genes. Both proteins were further studied with respect to their involvement in zinc homeostasis and invasion of the host. EF0759 contributed to intramacrophage survival by an as-yet unknown mechanism(s). EF1400, here renamed ZntAEf, is an ATPase with specificity for zinc and plays a role in dealing with several host defences, i.e. zinc overload, oxidative stress and lysozyme; it provides E. faecalis cells with the ability to survive inside macrophages. As these three host defence mechanisms are important at several sites in the host, i.e. inside macrophages and in saliva, this work suggested that ZntAEf constitutes a crucial E. faecalis defence mechanism that is likely to contribute to the ability of this bacterium to endure life inside its host.

  8. Theoretical study of phase behaviour of DLVO model for lysozyme and γ-crystalline aqueous electrolyte solutions

    Directory of Open Access Journals (Sweden)

    R. Melnyk


    Full Text Available Mean spherical approximation (MSA, second-order Barker-Henderson (BH perturbation theory and thermodynamic perturbation theory (TPT for associating fluids in combination with BH perturbation theory are applied to the study of the structural properties and phase behaviour of the Derjaguin-Landau-Verwey-Overbeek (DLVO model of lysozyme and γ-cristalline aqueous electrolyte solutions. Predictions of the MSA for the structure factors are in good agreement with the corresponding computer simulation predictions. The agreement between theoretical results for the liquid-gas phase diagram and the corresponding results of the experiment and computer simulation is less satisfactory, with predictions of the combined BH-TPT approach being the most accurate.

  9. Study of the binding between lysozyme and C10-TAB: determination and interpretation of the partial properties of protein and surfactant at infinite dilution. (United States)

    Morgado, Jorge; Aquino-Olivos, Marco Antonio; Martínez-Hernández, Ranulfo; Corea, Mónica; Grolier, Jean Pierre E; del Río, José Manuel


    This work examines the binding in aqueous solution, through the experimental determination of specific volumes and specific adiabatic compressibility coefficients, of decyltrimethylammonium bromide to lysozyme and to non-charged polymeric particles (which have been specially synthesized by emulsion polymerization). A method was developed to calculate the specific partial properties at infinite dilution and it was shown that a Gibbs-Duhem type equation holds at this limit for two solutes. With this equation, it is possible to relate the behavior of the partial properties along different binding types at a constant temperature. It was found that the first binding type, specific with high affinity, is related to a significant reduction of surfactant compressibility. The second binding type is accompanied by the unfolding of the protein and the third one is qualitatively identical to the binding of the surfactant to non-charged polymeric particles.

  10. Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization. (United States)

    Herrero, María Belén; Mandal, Arabinda; Digilio, Laura C; Coonrod, Scott A; Maier, Bernhard; Herr, John C


    This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.


    Directory of Open Access Journals (Sweden)

    L. Kurovskaya


    Full Text Available Purpose. To study the effect of рН values of the aquatic environment on the level of ectoparasite infestation, protein and lysozyme content in organs and serum of some cyprinid species in experimental conditions. Methodology. The objects of the study were yearlings of Cyprinus carpio, Carassius auratus gibelio and Pseudorasbora parva caught in ponds of fish farm “Nyvka” (Kiev region in spring. Fish were kept in experimental conditions at neutral pH water (6.8-7.2 and a temperature of 17-18оC. To study the changes in the level of fish parasite infestation at different pH values, we used carp yearlings, as an object, the most infected with parasites. Fish were placed in aquariums with water pH of 5.0-5.5 (slightly acidic environment and 8.5-9.0 (slightly alkaline environment for 5 days. Thereafter, the ectoparasites were counted on fish body surface and gills. The protein content in serum and tissue extracts of organs (liver, kidney, spleen of Carassius auratus gibelio and Pseudorasbora parva infected and uninfected ectoparasites, after holding them in slightly acidic or slightly alkaline environment for 8 days, was determined by Lowry’s method, while lysozyme content was determined by a diffusion method on agar. Findings. A comparative assessment of the number of ectoparasites (Ichthyophthirius multifiliis, Trichodina sp., Dactylogyrus sp., Gyrodactylus sp. on fish body surface and gills, the content of protein and lysozyme in serum and organs at different pH values of aquatic environment has been presented. It was demonstrated that the number of ectoparasites on fish body surface and gills was significantly reduced when keeping the fish in both slightly acidic and slightly alkaline environments. In infected Carassius auratus gibelio, a reduction in the protein and lysozyme content in liver, kidney and serum was observed only in the neutral pH environment compared to uninfected individuals. In the slightly acidic or slightly alkaline

  12. Diffusion coefficient of the protein in various crystallization solutions: The key to growing high-quality crystals in space (United States)

    Tanaka, Hiroaki; Takahashi, Sachiko; Yamanaka, Mari; Yoshizaki, Izumi; Sato, Masaru; Sano, Satoshi; Motohara, Moritoshi; Kobayashi, Tomoyuki; Yoshitomi, Susumu; Tanaka, Tetsuo; Fukuyama, Seijiro


    The diffusion coefficients of lysozyme and alpha-amylase were measured in the various polyethylene glycol (PEG) solutions. Obtained diffusion coefficients were studied with the viscosity coefficient of the solution. It was found that the diffusion process of the protein was suppressed with a factor of vγ, where ν is a relative viscosity coefficient of the PEG solution. The value of γ is -0.64 at PEG1500 for both proteins. The value increased to -0.48 at PEG8000 for lysozyme, while decreased to -0.72 for alpha-amylase. The equation of an approximate diffusion coefficient at certain PEG molecular weight and concentration was roughly obtained.

  13. Biophysical aspects of lysozyme adduct with monocrotophos. (United States)

    Amaraneni, Sreenivasa Rao; Kumar, Sudhir; Gourinath, Samudrala


    The present study on in vitro formation and characterization of lysozyme adduct with monocrotophos (MP) evaluates the potential of lysozyme to be used as a sensitive biomarker to monitor exposure levels to the commonly used organophosphorus pesticide monocrotophos. Crystallization of lysozyme protein adduct with monocrotophos was also undertaken to understand the adduct formation mechanism at a molecular level. The binding of organophosphorus pesticides to lysozyme is one of the key steps in their mutagenicity. The formation and structural characterization of lysozyme adduct with monocrotophos was done using MALDI-TOFMS, fluorescence, UV/Vis spectroscopy, circular dichroism, and X-ray diffraction studies. We report the crystal structure of lysozyme adduct with monocrotophos at 1.9 Å. It crystallized in the P43 space group with two monomers in one asymmetric unit having one molecule of monocrotophos bound to each protein chain. The results proved that the fluorescence quenching of lysozyme by monocrotophos is due to binding of monocrotophos with a tryptophan residue of lysozyme. Monocrotophos interacts most strongly with the Trp-108 and Asp-52 of lysozyme. The interactions of the monocrotophos molecule with the lysozyme suggest the formation of a stable adduct. In addition, the alteration of lysozyme secondary structure in the presence of monocrotophos was confirmed by circular dichroism and fluorescence inhibition of lysozyme by increasing monocrotophos and UV/Vis spectrophotometry. The formation of lysozyme adduct with monocrotophos was confirmed by MALDI-TOFMS.

  14. Pressure Effects on the Intermolecular Interaction Potential of Condensed Protein Solutions. (United States)

    Winter, Roland


    Knowledge of the intermolecular interaction potential of proteins as a function of their solution conditions is essential for understanding protein aggregation, crystallization, and the phase behavior of proteins in general. Here, we report on a combined small-angle X-ray scattering and liquid-state theoretical approach to study dense lysozyme solutions as a function of temperature and pressure, but also in the presence of salts and osmolytes of different nature. We show that the pressure-dependent interaction potential of lysozyme changes in a nonlinear fashion over a wide range of temperatures, salt and protein concentrations, indicating that changes of the bulk water structure mediate the pressure dependence of the intermolecular forces. We present also results on the effect of high hydrostatic pressure on the phase behavior of dense lysozyme solutions in the liquid-liquid phase-coexistence region. As also shown in this study, the application of pressure can be used to fine-tune the second virial coefficient of protein solutions, which can be used to control nucleation rates and hence protein crystallization, or to prevent protein aggregation. Moreover, these results are also important for understanding the hydration behavior of biological matter under extreme environmental conditions, and the high stability of dense protein solutions (as they occur intracellularly) in organisms thriving under hydrostatic pressure conditions such as in the deep sea, where pressures up to the 100 MPa-level are reached.



    L. Kurovskaya; G. Stril’ko


    Purpose. To study the effect of рН values of the aquatic environment on the level of ectoparasite infestation, protein and lysozyme content in organs and serum of some cyprinid species in experimental conditions. Methodology. The objects of the study were yearlings of Cyprinus carpio, Carassius auratus gibelio and Pseudorasbora parva caught in ponds of fish farm “Nyvka” (Kiev region) in spring. Fish were kept in experimental conditions at neutral pH water (6.8-7.2) and a temperature of 17...

  16. Prediction of thermodynamic instabilities of protein solutions from simple protein–protein interactions

    Energy Technology Data Exchange (ETDEWEB)

    D’Agostino, Tommaso [Dipartimento di Fisica, Università di Palermo, Via Archirafi 36, 90123 Palermo (Italy); Solana, José Ramón [Departamento de Física Aplicada, Universidad de Cantabria, 39005 Santander (Spain); Emanuele, Antonio, E-mail: [Dipartimento di Fisica, Università di Palermo, Via Archirafi 36, 90123 Palermo (Italy)


    Highlights: ► We propose a model of effective protein–protein interaction embedding solvent effects. ► A previous square-well model is enhanced by giving to the interaction a free energy character. ► The temperature dependence of the interaction is due to entropic effects of the solvent. ► The validity of the original SW model is extended to entropy driven phase transitions. ► We get good fits for lysozyme and haemoglobin spinodal data taken from literature. - Abstract: Statistical thermodynamics of protein solutions is often studied in terms of simple, microscopic models of particles interacting via pairwise potentials. Such modelling can reproduce the short range structure of protein solutions at equilibrium and predict thermodynamics instabilities of these systems. We introduce a square well model of effective protein–protein interaction that embeds the solvent’s action. We modify an existing model [45] by considering a well depth having an explicit dependence on temperature, i.e. an explicit free energy character, thus encompassing the statistically relevant configurations of solvent molecules around proteins. We choose protein solutions exhibiting demixing upon temperature decrease (lysozyme, enthalpy driven) and upon temperature increase (haemoglobin, entropy driven). We obtain satisfactory fits of spinodal curves for both the two proteins without adding any mean field term, thus extending the validity of the original model. Our results underline the solvent role in modulating or stretching the interaction potential.

  17. Measurements of Thermal Conductivity and Thermal Diffusivity of Hen Egg-White Lysozyme Crystals and Its Solution Using the Transient Short Hot Wire Method (United States)

    Fujiwara, Seiji; Maki, Syou; Maekawa, Ryunosuke; Tanaka, Seiichi; Hagiwara, Masayuki


    Protein crystals are an essentially important biological sample to advance the analysis of X-ray structure, but their thermophysical properties, especially thermal conductivity and thermal diffusivity, have not been studied sufficiently. This current situation can be attributed to various kinds of technical problems; e.g., the fragility of protein crystals and the difficulty of nucleation control. Ideally speaking, protein crystallization should be carried out under a " containerless condition" to eliminate any mechanical distortion of the crystals from the walls. To realize the condition, we have developed an original crystallization method by means of the magneto-Archimedes effect. In this paper, a transient short hot wire method was combined with the technique of magneto-Archimedes effect to realize simultaneous measurement of thermal conductivity and thermal diffusivity of hen egg-white lysozyme (HEWL) crystals. As the results, thermal conductivity and thermal diffusivity of HEWL crystals were found to be 0.410-0.438 \\hbox {W}\\cdot \\hbox {m}^{-1}\\cdot \\hbox {K}^{-1} and 3.77-5.18× 10^{-8} \\hbox {m}2\\cdot \\hbox {s}^{-1}, respectively. We clarified by the crystallizing process of HEWL that the crystals were magnetically levitated at the air-liquid interface and the short hot wire was completely buried into them as the crystals grew. We also measured the HEWL solution by the same methods. The thermal conductivity of the solution had almost the same value as that of water and had little dependency on the concentration of HEWL, but the thermal diffusivity was unclear.

  18. Role of the solvent in the dynamical transitions of proteins: The case of the lysozyme-water system (United States)

    Mallamace, Francesco; Chen, Sow-Hsin; Broccio, Matteo; Corsaro, Carmelo; Crupi, Vincenza; Majolino, Domenico; Venuti, Valentina; Baglioni, Piero; Fratini, Emiliano; Vannucci, Chiara; Stanley, H. Eugene


    We study the dynamics of hydration water in the protein lysozyme in the temperature range 180Kprotein hydration water. Below the first transition, at about 220K, the hydration water displays an unambiguous fragile-to-strong dynamic crossover, resulting in the loss of the protein conformational flexibility. Above the second transition, at about 346K, where the protein unfolds, the dynamics of the hydration water appears to be dominated by the non-hydrogen-bonded fraction of water molecules.

  19. Role of the solvent in the dynamical transitions of proteins: the case of the lysozyme-water system. (United States)

    Mallamace, Francesco; Chen, Sow-Hsin; Broccio, Matteo; Corsaro, Carmelo; Crupi, Vincenza; Majolino, Domenico; Venuti, Valentina; Baglioni, Piero; Fratini, Emiliano; Vannucci, Chiara; Stanley, H Eugene


    We study the dynamics of hydration water in the protein lysozyme in the temperature range 180 Kprotein hydration water. Below the first transition, at about 220 K, the hydration water displays an unambiguous fragile-to-strong dynamic crossover, resulting in the loss of the protein conformational flexibility. Above the second transition, at about 346 K, where the protein unfolds, the dynamics of the hydration water appears to be dominated by the non-hydrogen-bonded fraction of water molecules.

  20. New sub-family of lysozyme-like proteins shows no catalytic activity: crystallographic and biochemical study of STM3605 protein from Salmonella Typhimurium

    Energy Technology Data Exchange (ETDEWEB)

    Michalska, Karolina; Brown, Roslyn N.; Li, Hui; Jedrzejczak, Robert; Niemann, George; Heffron, Fred; Cort, John R.; Adkins, Joshua N.; Babnigg, Gyorgy; Joachimiak, Andrzej


    Phage viruses that infect prokaryotes integrate their genome into the host chromosome; thus, microbial genomes typically contain genetic remnants of both recent and ancient phage infections. Often phage genes occur in clusters of atypical G+C content that reflect integration of the foreign DNA. However, some phage genes occur in isolation without other phage gene neighbors, probably resulting from horizontal gene transfer. In these cases, the phage gene product is unlikely to function as a component of a mature phage particle, and instead may have been co-opted by the host for its own benefit. The product of one such gene from Salmonella enterica serovar Typhimurium, STM3605, encodes a protein with modest sequence similarity to phage-like lysozyme (N-acetylmuramidase) but appears to lack essential catalytic residues that are strictly conserved in all lysozymes. Close homologs in other bacteria share this characteristic. The structure of the STM3605 protein was characterized by X-ray crystallography, and functional assays showed that it is a stable, folded protein whose structure closely resembles lysozyme. However, this protein is unlikely to hydrolyze peptidoglycan. Instead, STM3605 is presumed to have evolved an alternative function because it shows some lytic activity and partitions to micelles.

  1. High-pressure protein crystallography of hen egg-white lysozyme

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Hiroyuki; Nagae, Takayuki [Nagoya University, Chikusa, Nagoya, Aichi 464-8603 (Japan); Watanabe, Nobuhisa, E-mail: [Nagoya University, Chikusa, Nagoya, Aichi 464-8603 (Japan); Nagoya University, Chikusa, Nagoya, Aichi 464-8603 (Japan)


    The crystal structure of hen egg-white lysozyme (HEWL) was analyzed under pressures of up to 950 MPa. The high pressure modified the conformation of the molecule and induced a novel phase transition in the tetragonal crystal of HEWL. Crystal structures of hen egg-white lysozyme (HEWL) determined under pressures ranging from ambient pressure to 950 MPa are presented. From 0.1 to 710 MPa, the molecular and internal cavity volumes are monotonically compressed. However, from 710 to 890 MPa the internal cavity volume remains almost constant. Moreover, as the pressure increases to 950 MPa, the tetragonal crystal of HEWL undergoes a phase transition from P4{sub 3}2{sub 1}2 to P4{sub 3}. Under high pressure, the crystal structure of the enzyme undergoes several local and global changes accompanied by changes in hydration structure. For example, water molecules penetrate into an internal cavity neighbouring the active site and induce an alternate conformation of one of the catalytic residues, Glu35. These phenomena have not been detected by conventional X-ray crystal structure analysis and might play an important role in the catalytic activity of HEWL.

  2. Proline is a protein solubilizing solute. (United States)

    Samuel, D; Kumar, T K; Jayaraman, G; Yang, P W; Yu, C


    The effect of proline on the prevention of trichloroacetic acid (TCA)-induced protein precipitation is studied. It is found that proline at high concentrations (> 4.0 M) completely prevents TCA-induced precipitation of hen egg white lysozyme. Other osmolytes such as ethylene glycol, glycerol and sucrose fail to prevent the TCA-induced precipitation of lysozyme. Viscosity and 1-anilino-8-naphthalene sulphonic acid binding experiments suggest that proline at high concentration forms an ordered supramolecular assembly. Proline is shown to increase the solubility of protein due to formation of such higher order assemblies. A model of the supra-molecular assembly of proline is proposed and a possible in vivo role of the increased levels of proline under water stress is discussed.

  3. Relationship Between Equilibrium Forms of Lysozyme Crystals and Precipitant Anions (United States)

    Nadarajah, Arunan


    Molecular forces, such as electrostatic, hydrophobic, van der Waals and steric forces, are known to be important in determining protein interactions. These forces are affected by the solution conditions and changing the pH, temperature or the ionic strength of the solution can sharply affect protein interactions. Several investigations of protein crystallization have shown that this process is also strongly dependent on solution conditions. As the ionic strength of the solution is increased, the initially soluble protein may either crystallize or form an amorphous precipitate at high ionic strengths. Studies done on the model protein hen egg white lysozyme have shown that different crystal forms can be easily and reproducibly obtained, depending primarily on the anion used to desolubilize the protein. In this study we employ pyranine to probe the effect of various anions on the water structure. Additionally, lysozyme crystallization was carried out at these conditions and the crystal form was determined by X-ray crystallography. The goal of the study was to understand the physico-chemical basis for the effect of changing the anion concentration on the equilibrium form of lysozyme crystals. It will also verify the hypothesis that the anions, by altering the bulk water structure in the crystallizing solutions, alter the surface energy of the between the crystal faces and the solution and, consequently, the equilibrium form of the crystals.

  4. Observance of polymorphic behaviour during dissolution of insulin and lysozyme

    Directory of Open Access Journals (Sweden)

    A. Bernardo


    Full Text Available Although protein crystallization is a unit operation with potentially high separation factors, it has not been widely used in industry. Protein crystallization studies and practices have hitherto been largely limited to crystallography protocols. Knowledge of the behaviour of protein in solution would help to overcome empiric limitations in protein crystallisation. Thus, dissolution of porcine insulin and hen egg white lysozyme was studied and an unusual variation in solute concentration, with a concentration peak for short dissolution times, was verified. Polymorphic behaviour of protein in solution was observed, which altered physical properties such as solubility.

  5. Dilatometric, refractometric and viscometric study of lysozyme-cation interaction. (United States)

    Abad, C; Trueba, M; Campos, A; Figueruelo, J E


    The interaction between hen egg-white lysozyme and Cu(II) or Co(II) cations has been studied by dilatometry, equilibrium dialysis-differential refractometry and viscometry at different metal cation concentrations. Delta V isotherms in copper and cobalt solutions have been obtained from dilatometry. Preferential adsorption parameters and specific viscosity have been determined from refractometric and viscosimetric measurements. It has been observed that this interaction produces structural alterations in lysozyme. The magnitude of these conformational changes depends on the metal ion and protein concentration. The results obtained using the three techniques are in good agreement.

  6. The association of lysozyme with casein

    NARCIS (Netherlands)

    Roos, de A.L.; Walstra, P.; Geurts, T.J.


    The association of hen eggs’ lysozyme with caseins was studied by using three casein substrates: (I) solutions of the various caseins, (II) artificially made casein micelles of various compositions and (III) caseins adsorbed onto soya-oil emulsion droplets. In solution, lysozyme associated most stro

  7. Electrostatic model for protein adsorption in ion-exchange chromatography and application to monoclonal antibodies, lysozyme and chymotrypsinogen A. (United States)

    Guélat, Bertrand; Ströhlein, Guido; Lattuada, Marco; Morbidelli, Massimo


    A model for the adsorption equilibrium of proteins in ion-exchange chromatography explicitly accounting for the effect of pH and salt concentration in the limit of highly diluted systems was developed. It is based on the use of DLVO theory to estimate the electrostatic interactions between the charged surface of the ion-exchanger and the proteins. The corresponding charge distributions were evaluated as a function of pH and salt concentration using a molecular approach. The model was verified for the adsorption equilibrium of lysozyme, chymotrypsinogen A and four industrial monoclonal antibodies on two strong cation-exchangers. The adsorption equilibrium constants of these proteins were determined experimentally at various pH values and salt concentrations and the model was fitted with a good agreement using three adjustable parameters for each protein in the whole range of experimental conditions. Despite the simplifications of the model regarding the geometry of the protein-ion-exchanger system, the physical meaning of the parameters was retained.

  8. Proteins identified from care solution extractions of silicone hydrogels. (United States)

    Emch, Andrew J; Nichols, Jason J


    The purpose of this study was to investigate the quantity and identify the proteins extracted from two different types of silicone hydrogel contact lenses by several multipurpose care solutions after 1 day of wear. Ten subjects were recruited to wear galyfilcon A lenses (Acuvue Advance, Vistakon) followed by lotrafilcon B lenses (O2 Optix, CIBA Vision) each for four consecutive days. Each day, subjects inserted a new pair of lenses for 8 h of wear after which both lenses were removed using forceps (lenses were not rubbed or rinsed after removal). Lenses were pooled in one of four commercially available care solutions for a 24-h soak followed by precipitation, resuspension in water, and quantification by Bradford assay and identification by mass spectrometry. Protein recovery from care solutions was as follows (quantities are in microg/lens): AQuify (galyfilcon A: 0.56, lotrafilcon B: 1.24), Complete MoisturePlus (galyfilcon A: 1.44, lotrafilcon B: 1.47), Opti-Free Express (galyfilcon A: 2.31, lotrafilcon B: 5.67), and ReNu MoistureLoc (galyfilcon A: 1.17, lotrafilcon B: 4.38). For each care solution, greater quantities of protein were removed from lotrafilcon B (3.19 +/- 2.19 microg/lens) than from galyfilcon A (1.37 +/- 0.72 microg/lens). Lactoferrin, lysozyme, and lipocalin were the most commonly identified, whereas various keratin compounds and other unique proteins were also detected. Opti-Free Express was consistently associated with the more efficient removal of proteins from these silicone hydrogels. More total protein was removed from lotrafilcon B than from galyfilcon A (approximately 2 x more protein) for all four care solutions, and 12 total unique protein species were recovered from galyfilcon A, whereas only 10 were recovered from lotrafilcon B. The higher quantities of protein extracted from lotrafilcon B may be due to stronger protein binding with this material and/or to differences in solution efficacy.

  9. Selectivity and localization of lysozyme uptake in contemporary hydrogel contact lens materials. (United States)

    Heynen, Miriam; Babaei Omali, Negar; Fadli, Zohra; Coles-Brennan, Chantal; Subbaraman, Lakshman N; Jones, Lyndon


    The purpose of this study was to investigate the early and selective uptake of lysozyme and the location of deposited lysozyme on contemporary hydrogel contact lens (CL) materials after exposure to an artificial tear solution (ATS) for 16 h. Seven different hydrogel CL materials [polymacon, omafilcon A, nelfilcon A, nesofilcon A, ocufilcon B, etafilcon A (Acuvue Moist), and etafilcon A (Acuvue Define)] were incubated in an ATS for various times. Total protein deposition was determined using a modified Bradford technique. Lysozyme, lactoferrin, and albumin deposition on CLs were determined using (125)I-radiolabeling method. A confocal laser scanning microscopy (CLSM) technique was utilized to map the location of lysozyme uptake in an asymmetric environment. All lens materials had significant amounts of lysozyme after 1 min of exposure to ATS. After 16 h of incubation, higher levels of total protein deposited on the two etafilcon A-based lenses (Moist and Define), followed by ocufilcon B and both were significantly higher than all other CLs tested (p = 0.0001). The two etafilcon A materials (Moist and Define) also deposited the highest amounts of lysozyme (514.8 ± 28.4 and 527.1 ± 14.7 μg/lens respectively) when compared to other test CLs (p = 0.0001). The CLSM technique revealed that the non-ionic CLs tended to have symmetric distribution of lysozyme throughout the lens materials, while the ionic CLs had an asymmetric distribution, with the highest concentration of lysozyme on and near the exposed surface. The quantity and nature of proteins deposited on CLs varies, depending upon the chemical composition of the material. Among the various lenses tested, etafilcon A deposited the highest amount of total protein, most of it represented by lysozyme, which was largely located near the surface of the lens.

  10. Competitive adsorption from mixed hen egg-white lysozyme/surfactant solutions at the air-water interface studied by tensiometry, ellipsometry, and surface dilational rheology. (United States)

    Alahverdjieva, V S; Grigoriev, D O; Fainerman, V B; Aksenenko, E V; Miller, R; Möhwald, H


    The competitive adsorption at the air-water interface from mixed adsorption layers of hen egg-white lysozyme with a non-ionic surfactant (C10DMPO) was studied and compared to the mixture with an ionic surfactant (SDS) using bubble and drop shape analysis tensiometry, ellipsometry, and surface dilational rheology. The set of equilibrium and kinetic data of the mixed solutions is described by a thermodynamic model developed recently. The theoretical description of the mixed system is based on the model parameters for the individual components.

  11. Complex coacervation of lysozyme and heparin

    DEFF Research Database (Denmark)

    van de Weert, Marco; Andersen, Mia Bendix; Frokjaer, Sven


    To characterize complex coacervates/flocculates of lysozyme and heparin in terms of binding stoichiometry and to determine the effect of complexation on protein structure and stability.......To characterize complex coacervates/flocculates of lysozyme and heparin in terms of binding stoichiometry and to determine the effect of complexation on protein structure and stability....

  12. Gold nanoparticles decorated with oligo(ethylene glycol) thiols: surface charges and interactions with proteins in solution. (United States)

    Schollbach, Moritz; Zhang, Fajun; Roosen-Runge, Felix; Skoda, Maximilian W A; Jacobs, Robert M J; Schreiber, Frank


    We have studied oligo(ethylene glycol) (OEG) thiol self-assembled monolayer (SAM) coated gold nanoparticles (AuOEG) and their interactions with proteins in solutions using electrophoretic and dynamic light scattering (ELS and DLS). The results are compared with poly(ethylene glycol) (PEG) thiol coated AuNPs (AuPEG). We show that both AuOEG and AuPEG particles carry a low net negative charge and are very stable (remaining so for more than one year), but long-term aging or dialysis can reduce the stability. If the decorated AuNPs are mixed with bovine serum albumin (BSA), both effective size and zeta-potential of the AuNPs remain unchanged, indicating no adsorption of BSA to the colloid surface. However, when mixed with lysozyme, zeta-potential values increase with protein concentrations and lead to a charge inversion, indicating adsorption of lysozyme to the colloid surface. The colloidal solutions of AuOEG become unstable near zero charge, indicated by a cluster peak in the DLS measurements. The AuPEG solutions show similar charge inversion upon addition of lysozyme, but the solutions are stable under all experimental conditions, presumably because of the strong steric effect of PEG. Washing the protein bound colloids by centrifugation can remove only part of the adsorbed lysozyme molecules indicating that a few proteins adsorb strongly to the colloids. The effective charge inversion and rather strongly bound lysozyme on the colloid surface may suggest that in addition to the charges formed at the SAM-water interface, there are defects on the surface of the colloid, which are accessible to the proteins. The results of this study of surface charge, and stability shed light on the interaction with proteins of SAM coated AuNPs and their applications. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Protein imprinted ionic liquid polymer on the surface of multiwall carbon nanotubes with high binding capacity for lysozyme. (United States)

    Yuan, Shifang; Deng, Qiliang; Fang, Guozhen; Wu, Jianhua; Li, Wangwang; Wang, Shuo


    In this research, ionic liquid as functional monomer to prepare molecularly imprinted polymers for protein recognition was for the first time demonstrated, in which, 1-vinyl-3-butylimidazolium chloride was selected as functional monomer, acrylamide as co-functional monomer and lysozyme (Lyz) as template protein to synthesize imprinted polymers on the surface of multiwall carbon nanotubes in aqueous medium. The results indicated that ionic liquid was helpful to improve binding capacity of imprinted polymers, which had a maximum binding capacity of 763.36 mg/g in the optimum adsorption conditions. The prepared imprinted polymers had a fast adsorption rate and a much higher adsorption capacity than the corresponding non-imprinted polymers, with the difference in adsorption capacity up to 258.31 mg/g. The obtained polymer was evaluated by Lyz, bovine serum albumin (BSA), bovine hemoglobin (BHb), equine myoglobin (MB) and cytochrome c (Cyt c). The selectivity factor (β) for Lyz/BSA, Lyz/Mb, Lyz/BHb, and Lyz/Cyt c were 7.17, 2.12, 1.76 and 1.57, respectively, indicating the imprinted polymers had a good selectivity. Easy preparation of the imprinted polymers as well as high ability and selectivity to adsorb template proteins makes this polymer attractive and broadly applicable in biomacromolecular separation, biotechnology and sensors.

  14. Mixed-mode chromatography integrated with high-performance liquid chromatography for protein analysis and separation: Using bovine serum albumin and lysozyme as the model target. (United States)

    Xia, Hai-Feng; Don, Bin-Bin; Zheng, Meng-Jie


    A type of mixed-mode chromatography was integrated with high-performance liquid chromatography for protein analysis and separation. The chromatographic behavior was tested using bovine serum albumin and lysozyme as model proteins. For the mixed-mode column, the silica beads were activated with γ-(2,3-epoxypropoxy)-propytrimethoxysilane and coupled with 4-mercaptopyridine as the functional ligand. The effects of pH, salt, and the organic solvent conditions of the mobile phase on the retention behavior were studied, which provided valuable clues for separation strategy. When eluted with a suitable pH gradient, salt concentration gradient, and acetonitrile content gradient, the separation behavior of bovine serum albumin and lysozyme could be controlled by altering the conditions of the mobile phase. The results indicated this type of chromatography might be a useful method for protein analysis and separation.

  15. A new family of lysozyme inhibitors contributing to lysozyme tolerance in gram-negative bacteria.

    Directory of Open Access Journals (Sweden)

    Lien Callewaert


    Full Text Available Lysozymes are ancient and important components of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall polymer. Bacteria engaging in commensal or pathogenic interactions with an animal host have evolved various strategies to evade this bactericidal enzyme, one recently proposed strategy being the production of lysozyme inhibitors. We here report the discovery of a novel family of bacterial lysozyme inhibitors with widespread homologs in gram-negative bacteria. First, a lysozyme inhibitor was isolated by affinity chromatography from a periplasmic extract of Salmonella Enteritidis, identified by mass spectrometry and correspondingly designated as PliC (periplasmic lysozyme inhibitor of c-type lysozyme. A pliC knock-out mutant no longer produced lysozyme inhibitory activity and showed increased lysozyme sensitivity in the presence of the outer membrane permeabilizing protein lactoferrin. PliC lacks similarity with the previously described Escherichia coli lysozyme inhibitor Ivy, but is related to a group of proteins with a common conserved COG3895 domain, some of them predicted to be lipoproteins. No function has yet been assigned to these proteins, although they are widely spread among the Proteobacteria. We demonstrate that at least two representatives of this group, MliC (membrane bound lysozyme inhibitor of c-type lysozyme of E. coli and Pseudomonas aeruginosa, also possess lysozyme inhibitory activity and confer increased lysozyme tolerance upon expression in E. coli. Interestingly, mliC of Salmonella Typhi was picked up earlier in a screen for genes induced during residence in macrophages, and knockout of mliC was shown to reduce macrophage survival of S. Typhi. Based on these observations, we suggest that the COG3895 domain is a common feature of a novel and widespread family of bacterial lysozyme inhibitors in gram-negative bacteria that may function as colonization or virulence factors in bacteria

  16. Evolution of the mammalian lysozyme gene family

    Directory of Open Access Journals (Sweden)

    Biegel Jason M


    Full Text Available Abstract Background Lysozyme c (chicken-type lysozyme has an important role in host defense, and has been extensively studied as a model in molecular biology, enzymology, protein chemistry, and crystallography. Traditionally, lysozyme c has been considered to be part of a small family that includes genes for two other proteins, lactalbumin, which is found only in mammals, and calcium-binding lysozyme, which is found in only a few species of birds and mammals. More recently, additional testes-expressed members of this family have been identified in human and mouse, suggesting that the mammalian lysozyme gene family is larger than previously known. Results Here we characterize the extent and diversity of the lysozyme gene family in the genomes of phylogenetically diverse mammals, and show that this family contains at least eight different genes that likely duplicated prior to the diversification of extant mammals. These duplicated genes have largely been maintained, both in intron-exon structure and in genomic context, throughout mammalian evolution. Conclusions The mammalian lysozyme gene family is much larger than previously appreciated and consists of at least eight distinct genes scattered around the genome. Since the lysozyme c and lactalbumin proteins have acquired very different functions during evolution, it is likely that many of the other members of the lysozyme-like family will also have diverse and unexpected biological properties.

  17. Relaxation dynamics of a protein solution investigated by dielectric spectroscopy. (United States)

    Wolf, M; Gulich, R; Lunkenheimer, P; Loidl, A


    In the present work, we provide a dielectric study on two differently concentrated aqueous lysozyme solutions in the frequency range from 1MHz to 40GHz and for temperatures from 275 to 330K. We analyze the three dispersion regions, commonly found in protein solutions, usually termed β-, γ-, and δ-relaxations. The β-relaxation, occurring in the frequency range around 10MHz and the γ-relaxation around 20GHz (at room temperature) can be attributed to the rotation of the polar protein molecules in their aqueous medium and the reorientational motion of the free water molecules, respectively. The nature of the δ-relaxation, which is often ascribed to the motion of bound water molecules, is not yet fully understood. Here we provide data on the temperature dependence of the relaxation times and relaxation strengths of all three detected processes and on the dc conductivity arising from ionic charge transport. The temperature dependences of the β- and γ-relaxations are closely correlated. We found a significant temperature dependence of the dipole moment of the protein, indicating conformational changes. Moreover we find a breakdown of the Debye-Stokes-Einstein relation in this protein solution, i.e., the dc conductivity is not completely governed by the mobility of the solvent molecules. Instead it seems that the dc conductivity is closely connected to the hydration shell dynamics.

  18. Effect of surface energy of solid surfaces on the micro- and macroscopic properties of adsorbed BSA and lysozyme. (United States)

    Sharma, Indu; Pattanayek, Sudip K


    The surface energy, a macroscopic property, depends on the chemical functionality and micro- and macroscopic roughness of the surface. The adsorption of two widely used proteins bovine serum albumin (BSA) and lysozyme on surfaces of four different chemical functionalities were done to find out the interrelation between macroscopic and microscopic properties. We have observed the secondary structure of protein after its adsorption. In addition, we observed the variation of surface energy of proteins due to variation in adsorption time, change in protein concentration and effect of a mixture of proteins. Surfaces of three different chemical functionalities namely, amine, hydroxyl and octyl were obtained through self-assembled monolayer on silica surfaces and were tested for responses towards adsorption of lysozyme and BSA. The adsorbed lysozyme has higher surface energy than the adsorbed BSA on amine and octyl surfaces. On hydroxyl functional surface, the surface energy due to the adsorbed lysozyme or BSA increases slowly with time. The surface energy of the adsorbed protein increases gradually with increasing protein concentration on hydrophobic surfaces. On hydrophilic surfaces, with increasing BSA concentration in bulk solution, the surface energy of the adsorbed protein on GPTMS and amine surfaces is maximum at 1μM concentration. During the adsorption from a mixture of BSA and lysozyme on octyl surface, first lysozyme adsorbs and subsequent BSA adsorption leads to a high surface energy. Copyright © 2016. Published by Elsevier B.V.

  19. Control of electrostatic interactions between F-actin and genetically modified lysozyme in aqueous media

    Energy Technology Data Exchange (ETDEWEB)

    Sanders, Lori K.; Xian, Wujing; Guaqueta, Camilo; Strohman, Michael J.; Vrasich, Chuck R.; Luijten, Erik; Wong, Gerard C.L. (UIUC)


    The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin-lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin-lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin-lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity.

  20. Control of Electrostatic Interactions Between F-Actin And Genetically Modified Lysozyme in Aqueous Media

    Energy Technology Data Exchange (ETDEWEB)

    Sanders, L.K.; Xian, W.; Guaqueta, C.; Strohman, M.; Vrasich, C.R.; Luijten, E.; Wong, G.C.L.


    The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin-lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin-lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin-lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity.

  1. Effect of ethanol as a co-solvent on the aerosol performance and stability of spray-dried lysozyme

    DEFF Research Database (Denmark)

    Ji, Shuying; Thulstrup, Peter Waaben; Mu, Huiling


    In the spray drying process, organic solvents can be added to facilitate drying, accommodate certain functional excipients, and modify the final particle characteristics. In this study, lysozyme was used as a model pharmaceutical protein to study the effect of ethanol as a co-solvent on the stabi......In the spray drying process, organic solvents can be added to facilitate drying, accommodate certain functional excipients, and modify the final particle characteristics. In this study, lysozyme was used as a model pharmaceutical protein to study the effect of ethanol as a co......-solvent on the stability and aerosol performance of spray-dried protein. Lysozyme was dissolved in solutions with various ratios of ethanol and water, and subsequently spray-dried. A change from spherical particles into wrinkled and folded particles was observed upon increasing the ratio of ethanol in the feed....... The aerosol performance of the spray-dried lysozyme from ethanol-water solution was improved compared to that from pure water. The conformation of lysozyme in the ethanol-water solution and spray dried powder was altered, but the native structure of lysozyme was restored upon reconstitution in water after...

  2. Effect of Cholesterol on the Properties of Spray-Dried Lysozyme-Loaded Liposomal Powders


    Charnvanich, Dusadee; Vardhanabhuti, Nontima; Kulvanich, Poj


    The influence of cholesterol (Chol) in the liposomal bilayer on the properties of inhalable protein-loaded liposomal powders prepared by spray-drying technique was investigated. Lysozyme (LSZ) was used as a model protein. Feed solution for spray drying was prepared by direct mixing of aqueous solution of LSZ with mannitol solution and empty liposome dispersions composed of hydrogenated phosphatidylcholine and Chol at various molar ratios. The spray-dried powders were characterized with respec...

  3. Amphiphilic copolymers reduce aggregation of unfolded lysozyme more effectively than polyethylene glycol (United States)

    Chin, Jaemin; Mustafi, Devkumar; Poellmann, Michael J.; Lee, Raphael C.


    Certain amphiphilic block copolymers are known to prevent aggregation of unfolded proteins. To better understand the mechanism of this effect, the optical properties of heat-denatured and dithiothreitol reduced lysozyme were evaluated with respect to controls using UV–Vis spectroscopy, transmission electron microscopy (TEM) and circular dichroism (CD) measurements. Then, the effects of adding Polyethylene Glycol (8000 Da), the triblock surfactant Poloxamer 188 (P188), and the tetrablock copolymer Tetronic 1107 (T1107) to the lysozyme solution were compared. Overall, T1107 was found to be more effective than P188 in inhibiting aggregation, while PEG exhibited no efficacy. TEM imaging of heat-denatured and reduced lysozymes revealed spherical aggregates with on average 250–450 nm diameter. Using CD, more soluble lysozyme was recovered with T1107 than P188 with β-sheet secondary structure. The greater effectiveness of the larger T1107 in preventing aggregation of unfolded lysozyme than the smaller P188 and PEG points to steric hindrance at play; signifying the importance of size match between the hydrophobic region of denatured protein and that of amphiphilic copolymers. Thus, our results corroborate that certain multi-block copolymers are effective in preventing heat-induced aggregation of reduced lysozymes and future studies warrant more detailed focus on specific applications of these copolymers.

  4. Enterococcus faecalis zinc-responsive proteins mediate bacterial defence against zinc overload, lysozyme and oxidative stress

    NARCIS (Netherlands)

    C. Abrantes, Marta; Kok, Jan; de Fatima Silva Lopes, Maria


    Two Enterococcus faecalis genes encoding the P-type ATPase EF1400 and the putative SapB protein EF0759 were previously shown to be strongly upregulated in the presence of high concentrations of zinc. In the present work, we showed that a Zn(2+)-responsive DNA-binding motif (zim) is present in the pr

  5. Solutal Convection Around Growing Protein Crystal and Diffusional Purification in Space (United States)

    Lee, Chun P.; Chernov, Alexander A.


    At least some protein crystals were found to preferentially trap microheterogeneous impurities. The latter are, for example, dimmer molecules of the crystallizing proteines (e.g. ferritin, lysozyme), or the regular molecules on which surfaces small molecules or ions are adsorbed (e.g. acetilated lysozyme) and modi@ molecular charge. Impurities may induce lattice defects and deteriorate structural resolution. Distribution of impurities between mother solution and gorwing crystal is defined by two interrelated distribution coefficients: kappa = rho(sup c, sub 2) and K = (rho(sup c, sub 2)/rho(sup c, sub 1)/rho(sub 2)/rho(sub 1). Here, rho(sub 2), rho(sub 1) and rho(sup c, sub 2) are densities of impurity (2) and regular protein (1) in solution at the growing interface and within the crystal ("c"). For the microheterogeneous impurities studied, K approx. = 2 - 4, so that kappa approx. - 10(exp 2) - 10(exp 3), since K = kappa (rho(sub 1)/rho(sup c, sub 1) and protein solubility ratio rho(sub 1)/rho(sub=p c, sub 2) much less than 1. Therefore, a crystal growing in absence of convection purifies mother solution around itself, grows cleaner and, probably, more perfect. If convection is present, the solution flow permanently brings new impurities to the crystal. This work theoretically addressed two subjects: 1) onset of convection, 2) distribution of impurities.

  6. Synthesis of hydrophobic nanoparticles for real-time lysozyme detection using surface plasmon resonance sensor. (United States)

    Saylan, Yeşeren; Yılmaz, Fatma; Derazshamshir, Ali; Yılmaz, Erkut; Denizli, Adil


    Diagnostic biomarkers such as proteins and enzymes are generally hard to detect because of the low abundance in biological fluids. To solve this problem, the advantages of surface plasmon resonance (SPR) and nanomaterial technologies have been combined. The SPR sensors are easy to prepare, no requirement of labelling and can be detected in real time. In addition, they have high specificity and sensitivity with low cost. The nanomaterials have also crucial functions such as efficiency improvement, selectivity, and sensitivity of the detection systems. In this report, an SPR-based sensor is developed to detect lysozyme with hydrophobic poly (N-methacryloyl-(L)-phenylalanine) (PMAPA) nanoparticles. The SPR sensor was first characterized by attenuated total reflection-Fourier transform infrared, atomic force microscope, and water contact angle measurements and performed with aqueous lysozyme solutions. Various concentrations of lysozyme solution were used to calculate kinetic and affinity coefficients. The equilibrium and adsorption isotherm models of interactions between lysozyme solutions and SPR sensor were determined and the maximum reflection, association, and dissociation constants were calculated by Langmuir model as 4.87, 0.019 nM(-1) , and 54 nM, respectively. The selectivity studies of SPR sensor were investigated with competitive agents, hemoglobin, and myoglobin. Also, the SPR sensor was used four times in adsorption/desorption/recovery cycles and results showed that, the combination of optical SPR sensor with hydrophobic ionizable PMAPA nanoparticles in one mode enabled the detection of lysozyme molecule with high accuracy, good sensivity, real-time, label-free, and a low-detection limit of 0.66 nM from lysozyme solutions. Lysozyme detection in a real sample was performed by using chicken egg white to evaluate interfering molecules present in the medium.


    NARCIS (Netherlands)



    Background: Hevamine is a member of one of several families of plant chitinases and lysozymes that are important for plant defence against pathogenic bacteria and fungi. The enzyme can hydrolyze the linear polysaccharide chains of chitin and peptidoglycan. A full understanding of the structure/funct

  8. Self-assembly dynamics for the transition of a globular aggregate to a fibril network of lysozyme proteins via a coarse-grained Monte Carlo simulation (United States)

    Pandey, R. B.; Farmer, B. L.; Gerstman, Bernard S.


    The self-organizing dynamics of lysozymes (an amyloid protein with 148 residues) with different numbers of protein chains, Nc = 1,5,10, and 15 (concentration 0.004 - 0.063) is studied by a coarse-grained Monte Carlo simulation with knowledge-based residue-residue interactions. The dynamics of an isolated lysozyme (Nc = 1) is ultra-slow (quasi-static) at low temperatures and becomes diffusive asymptotically on raising the temperature. In contrast, the presence of interacting proteins leads to concentration induced protein diffusion at low temperatures and concentration-tempering sub-diffusion at high temperatures. Variation of the radius of gyration of the protein with temperature shows a systematic transition from a globular structure (at low T) to a random coil (high T) conformation when the proteins are isolated. The crossover from globular to random coil becomes sharper upon increasing the protein concentration (i.e. with Nc = 5,10), with larger Rg at higher temperatures and concentration; Rg becomes smaller on adding more protein chains (e.g. Nc = 15) a non-monotonic response to protein concentration. Analysis of the structure factor (S(q)) provides an estimate of the effective dimension (D ≥ 3, globular conformation at low temperature, and D ˜ 1.7, random coil, at high temperatures) of the isolated protein. With many interacting proteins, the morphology of the self-assembly varies with scale, i.e. at the low temperature (T = 0.015), D ˜ 2.9 on the scale comparable to the radius of gyration of the protein, and D ˜ 2.3 at the large scale over the entire sample. The global network of fibrils appears at high temperature (T = 0.021) with D ˜ 1.7 (i.e. a random coil morphology at large scale) involving tenuous distribution of micro-globules (at small scales).

  9. Self-assembly dynamics for the transition of a globular aggregate to a fibril network of lysozyme proteins via a coarse-grained Monte Carlo simulation

    Directory of Open Access Journals (Sweden)

    R. B. Pandey


    Full Text Available The self-organizing dynamics of lysozymes (an amyloid protein with 148 residues with different numbers of protein chains, Nc = 1,5,10, and 15 (concentration 0.004 – 0.063 is studied by a coarse-grained Monte Carlo simulation with knowledge-based residue-residue interactions. The dynamics of an isolated lysozyme (Nc = 1 is ultra-slow (quasi-static at low temperatures and becomes diffusive asymptotically on raising the temperature. In contrast, the presence of interacting proteins leads to concentration induced protein diffusion at low temperatures and concentration-tempering sub-diffusion at high temperatures. Variation of the radius of gyration of the protein with temperature shows a systematic transition from a globular structure (at low T to a random coil (high T conformation when the proteins are isolated. The crossover from globular to random coil becomes sharper upon increasing the protein concentration (i.e. with Nc = 5,10, with larger Rg at higher temperatures and concentration; Rg becomes smaller on adding more protein chains (e.g. Nc = 15 a non-monotonic response to protein concentration. Analysis of the structure factor (S(q provides an estimate of the effective dimension (D ≥ 3, globular conformation at low temperature, and D ∼ 1.7, random coil, at high temperatures of the isolated protein. With many interacting proteins, the morphology of the self-assembly varies with scale, i.e. at the low temperature (T = 0.015, D ∼ 2.9 on the scale comparable to the radius of gyration of the protein, and D ∼ 2.3 at the large scale over the entire sample. The global network of fibrils appears at high temperature (T = 0.021 with D ∼ 1.7 (i.e. a random coil morphology at large scale involving tenuous distribution of micro-globules (at small scales.

  10. Acetylated Lysozyme as Impurity in Lysozyme Crystals: Constant Distribution Coefficient (United States)

    Thomas, B. R.; Chernov, A. A.


    Hen egg white lysozyme (HEWL) was acetylated to modify molecular charge keeping the molecular size and weight nearly constant. Two derivatives, A and B, more and less acetylated, respectively, were obtained, separated, purified and added to the solution from which crystals of tetragonal HEWL crystals were grown. Amounts of the A or B impurities added were 0.76, 0.38 and 0.1 milligram per millimeter while HEWL concentration were 20, 30 and 40 milligram per milliliter. The crystals grown in 18 experiments for each impurity were dissolved and quantities of A or B additives in these crystals were analyzed by cation exchange high performance liquid chromatography. All the data for each set of 18 samples with the different impurity and regular HEWL concentrations is well described by one distribution coefficient K = 2.15 plus or minus 0.13 for A and K = 3.42 plus or minus 0.25 for B. The observed independence of the distribution coefficient on both the impurity concentration and supersaturation is explained by the dilution model described in this paper. It shows that impurity adsorption and incorporation rate is proportional to the impurity concentration and that the growth rate is proportional to the crystallizing protein in solution. With the kinetic coefficient for crystallization, beta = 5.10(exp -7) centimeters per second, the frequency at which an impurity molecule near the growing interface irreversibly joins a molecular site on the crystal was found to be 3 1 per second, much higher than the average frequency for crystal molecules. For best quality protein crystals it is better to have low microheterogeneous protein impurity concentration and high supers aturation.


    Institute of Scientific and Technical Information of China (English)

    Jing-jing Liu; Yun-feng Yan; Ping Yao


    Butyl modified poly(allylamine)s with butyl substitution degrees of 15% to 70% were prepared. The polymers show pH sensitive property and lower critical solution temperature (LCST) behavior. The LCST appears at lower temperature, lower pH and lower polymer concentration for the polymer with higher butylated degree. The binding of native lysozyme with the polymers depends on the hydrophobicity of the polymers at the pH range that the protein and the polymer carry the same positive charges. The increase of polymer hydrophobicity can increase the binding with lysozyme, but the self-aggregation of the polymer decreases the binding. The bound lysozyme molecules can recover their native activity completely after the dissociation of the complexes. Compared with native lysozyme, the denatured one which exposes the hydrophobic residues can increase the binding with the polymer and form stable complex nanoparticles.

  12. The molecular interaction of a protein in highly concentrated solution investigated by Raman spectroscopy. (United States)

    Ota, Chikashi; Noguchi, Shintaro; Tsumoto, Kouhei


    We used Raman spectroscopy to investigate the structure and interactions of lysozyme molecules in solution over a wide range of concentrations (2.5-300 mg ml(-1)). No changes in the amide-I band were observed as the concentration was increased, but the width of the Trp band at 1555 cm(-1) and the ratios of the intensities of the Tyr bands at 856 and 837 cm(-1), the Trp bands at 870 and 877 cm(-1), and the bands at 2940 (CH stretching) and 3420 cm(-1) (OH stretching) changed as the concentration was changed. These results reveal that although the distance between lysozyme molecules changed by more than an order of magnitude over the tested concentration range, the secondary structure of the protein did not change. The changes in the molecular interactions occurred in a stepwise process as the order of magnitude of the distance between molecules changed. These results suggest that Raman bands can be used as markers to investigate the behavior of high-concentration solutions of proteins and that the use of Raman spectroscopy will lead to progress in our understanding not only of the basic science of protein behavior under concentrated (i.e., crowded) conditions but also of practical processes involving proteins, such as in the field of biopharmaceuticals. © 2014 Wiley Periodicals, Inc.

  13. Effects of Convective Solute and Impurity Transport in Protein Crystal Growth (United States)

    Vekilov, Peter G.; Thomas, Bill R.; Rosenberger, Franz


    High-resolution optical interferometry was used to investigate the effects of forced solution convection on the crystal growth kinetics of the model protein lysozyme. Most experiments were conducted with 99.99% pure protein solutions. To study impurity effects, approx. 1% of lysozyme dimer (covalently bound) was added in some cases. We show that the unsteady kinetics, corresponding to bunching of growth steps, can be characterized by the Fourier components of time traces of the growth rate. Specific Fourier spectra are uniquely determined by the solution conditions (composition, temperature, and flow rate) and the growth layer source activity. We found that the average step velocity and growth rate increase by approx. I0% with increasing flow rate, as a result of the enhanced solute supply to the interface. More importantly, faster convective transport results in lower fluctuation amplitudes. This observation supports our rationale for system-dependent effects of transport on the structural perfection of protein crystals. We also found that solution flow rates greater than 500 microns/s result in stronger fluctuations while the average growth rate is decreased. This can lead to growth cessation at low supersaturations. With the intentionally contaminated solutions, these undesirable phenomena occurred at about half the flow rates required in pure solutions. Thus, we conclude that they are due to enhanced convective supply of impurities that are incorporated into the crystal during growth. Furthermore, we found that the impurity effects are reduced at higher crystal growth rates. Since the exposure time of terraces is inversely proportional to the growth rate, this observation suggests that the increased kinetics instability results from impurity adsorption on the interface. Finally, we provide evidence relating earlier observations of "slow protein crystal growth kinetics" to step bunch formation in response to nonsteady step generation.

  14. Hexafluoroisopropanol-induced catanionic-surfactants-based coacervate extraction for analysis of lysozyme. (United States)

    Xu, Jia; Niu, Manli; Xiao, Yuxiu


    A coacervate extraction method, based on hexafluoroisopropanol (HFIP)-induced catanionic surfactants and coupled with a back-extraction procedure, was developed for separation and purification of proteins, using sodium dodecyl sulfate (SDS) and dodecyltrimethyl ammonium bromide (DTAB) as representative catanionic surfactants and lysozyme as a model protein. After the coacervate extraction and back extraction, the obtained lysozyme solutions were examined in terms of quantitative analysis by capillary electrophoresis, bacteriolytic activity, and circular dichroism (CD). The effects of several parameters including back-extraction solvent, HFIP content, total surfactant concentration, and SDS/DTAB molar ratio were investigated in detail on the extraction efficiency and activity of lysozyme. Under the optimized extraction conditions (66 mM KH2PO4 buffer with pH 6.2 as back-extraction solvent, SDS/DTAB molar ratio = 1:1 mol/mol, total surfactant concentration = 30 mM, HIFP concentration = 8 % v/v), the extraction recovery was 89.8 % (±4.7, n = 3), limit of detection was 2.2 (±0.3, n = 3) μg mL(-1), and meanwhile nearly 65 % of native lysozyme activity was retained. In addition, the activity and CD assays showed that SDS/DTAB molar ratio had a significant influence on the activity and structure of lysozyme after extraction. The DTAB-rich extraction systems, in which the DTAB mole fraction was equal to or larger than 70 %, could keep the activity and structure of lysozyme almost in the native state. Graphical Abstract Procedure of HFIP-induced SDS/DTAB coacervate extraction and back extraction of lysozyme.

  15. Lysozyme gelation in mixtures of tetramethylurea with protic solvents: Use of solvatochromic indicators to probe medium microstructure and solute solvent interactions (United States)

    da Silva, Marcelo A.; El Seoud, Omar A.; Arêas, Elizabeth P. G.


    This work investigated the relationship between the structure of binary mixtures of tetramethylurea and protic solvents and their capacity to induce lysozyme gelation. In order to get an insight into the mechanism of gel formation, the solvatochromic behavior of zwitterionic probes, employed as simple models for the protein, was investigated. We studied two probes of similar p Ka's, but different hydrophobic character, namely 2,6-diphenyl-4-(2,4,6-triphenylpyridinium-1-yl) phenolate, RB, and 4-[2-(1-methylpyridinium-4-yl) ethenyl] phenolate, MC. The protic solvents used included water, 1-propanol and 2- n-butoxyethanol in the temperature range from 10 to 60 °C, and methanol, from 10 to 40 °C. In all cases, the dependence of the empirical solvent polarity parameter, ET, on mixture composition was non-ideal with negative deviation for TMU-water and positive deviation for TMU-organic solvent. For all binary mixtures, the deviation from linearity decreased as a function of increasing the temperature. In TMU/alcohol, the effect became more pronounced with increasing alcohol hydrophobicity.

  16. Equilibrium and dynamic interfacial properties of protein/ionic-liquid-type surfactant solutions at the decane/water interface. (United States)

    Cao, Chong; Lei, Jinmei; Zhang, Lu; Du, Feng-Pei


    The interfacial behavior of β-casein and lysozyme solutions has been investigated in the presence of an ionic liquid-type imidazolium surfactant ([C16mim]Br) at the decane/water interface. The dynamic dilational properties of the protein/surfactant solutions are investigated by the oscillating drop method and interfacial tension relaxation method. The interfacial tension isotherms for the mixed adsorption layers indicate that the increased addition of [C16mim]Br to a pure protein changes the properties of the complex formed at the decane/water interface. Whereas the interfacial tension data of the protein/surfactant mixed layers do not clearly show differences with changing bulk composition, the dilational rheology provides undoubted evidence that the structure and, in particular, the dynamics of the adsorbed layers depend on the bulk surfactant concentration. The experiment data for β-casein/[C16mim]Br solutions indicate that at higher bulk [C16mim]Br concentrations, β-casein in the interfacial layer is subject to conformational changes, where it gives space to [C16mim]Br molecules in the form of coadsorb rather than replacement; in contrast, in lysozyme/[C16mim]Br solutions some lysozyme molecules desorb from the interface due to the competitive adsorption of free [C16mim]Br molecules. Experimental results related to the interfacial dilational properties of the protein/surfactant solutions show that the dilational modulus turns out to be more sensitive to the conformation of protein/surfactant mixture at the liquid interface than the interfacial tension.

  17. Antimicrobial activity and synergism of lactoferrin and lysozyme against cariogenic microorganisms. (United States)

    de Andrade, Flaviana Bombarda; de Oliveira, Jair Caetano; Yoshie, Marjorie Takei; Guimarães, Bruno Martini; Gonçalves, Rafael Braga; Schwarcz, Waleska Dias


    The present study evaluated the antimicrobial in vitro effects of the salivary proteins lactoferrin and lysozyme on microorganisms involved in the carious process, obtaining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Streptococcus mutans (ATCC 25175) and Lactobacillus casei (ATCC 7469) were submitted to broth macrodilution of lysozyme at 80 mg/mL and lactoferrin at 200 mg/mL. The tubes were read in a spectrophotometer after they had been incubated at 37 °C for 18 h, in a carbon dioxide chamber, in order to read the MIC. A new subculture was carried on agar plates to obtain the MBC. The agar diffusion method was also tested, using BHI agar with 100 µL of the standardized microbial inocula. Filter-paper disks soaked in 10 µL of the solutions lactoferrin (200 µg/mL) and lysozyme (80 µg/mL) were placed on the agar surface. Inhibition halos were not observed on the plates, showing the absence of the antimicrobial effects of these proteins in this method. The bactericidal and bacteriostatic effects of lysozyme on L. casei were 50.3 mg/mL and 43.1 mg/mL respectively. The bactericidal and bacteriostatic effects on S. mutans were 68.5 mg/mL and 58.7 mg/mL. Lactoferrin did not induce any inhibitory effects on any microorganism, even in the concentration of 200 mg/mL. There was not a synergic antimicrobial effect of proteins, when they were tested together, even in the concentration of 42.8 mg/mL of lysozyme and 114 mg/mL of lactoferrin (the highest values evaluated). S. mutans and L. casei were only inhibited by lysozyme, not affected by lactoferrin and by the synergic use of both proteins.

  18. Terahertz underdamped vibrational motion governs protein-ligand binding in solution. (United States)

    Turton, David A; Senn, Hans Martin; Harwood, Thomas; Lapthorn, Adrian J; Ellis, Elizabeth M; Wynne, Klaas


    Low-frequency collective vibrational modes in proteins have been proposed as being responsible for efficiently directing biochemical reactions and biological energy transport. However, evidence of the existence of delocalized vibrational modes is scarce and proof of their involvement in biological function absent. Here we apply extremely sensitive femtosecond optical Kerr-effect spectroscopy to study the depolarized Raman spectra of lysozyme and its complex with the inhibitor triacetylchitotriose in solution. Underdamped delocalized vibrational modes in the terahertz frequency domain are identified and shown to blue-shift and strengthen upon inhibitor binding. This demonstrates that the ligand-binding coordinate in proteins is underdamped and not simply solvent-controlled as previously assumed. The presence of such underdamped delocalized modes in proteins may have significant implications for the understanding of the efficiency of ligand binding and protein-molecule interactions, and has wider implications for biochemical reactivity and biological function.

  19. The Antimicrobial Peptide Lysozyme Is Induced after Multiple Trauma

    Directory of Open Access Journals (Sweden)

    Tim Klüter


    Full Text Available The antimicrobial peptide lysozyme is an important factor of innate immunity and exerts high potential of antibacterial activity. In the present study we evaluated the lysozyme expression in serum of multiple injured patients and subsequently analyzed their possible sources and signaling pathways. Expression of lysozyme was examined in blood samples of multiple trauma patients from the day of trauma until 14 days after trauma by ELISA. To investigate major sources of lysozyme, its expression and regulation in serum samples, different blood cells, and tissue samples were analysed by ELISA and real-time PCR. Neutrophils and hepatocytes were stimulated with cytokines and supernatant of Staphylococcus aureus. The present study demonstrates the induction and release of lysozyme in serum of multiple injured patients. The highest lysozyme expression of all tested cells and tissues was detected in neutrophils. Stimulation with trauma-related factors such as interleukin-6 and S. aureus induced lysozyme expression. Liver tissue samples of patients without trauma show little lysozyme expression compared to neutrophils. After stimulation with bacterial fragments, lysozyme expression of hepatocytes is upregulated significantly. Toll-like receptor 2, a classic receptor of Gram-positive bacterial protein, was detected as a possible target for lysozyme induction.

  20. Viscoelastic Phase Separation of Protein Solutions (United States)

    Tanaka, Hajime; Nishikawa, Yuya


    In addition to the known behavior of normal phase separation and gelation, we report novel phase-separation behavior of protein solutions as their intermediate case. A network structure of the protein-rich phase may be formed even if it is the minority phase, contrary to the conventional wisdom. This behavior is characteristic of viscoelastic phase separation found in polymer solutions. This kinetic pathway may play crucial roles in the complex phase ordering of protein solutions, in particular, protein network formation in biological systems and foods.

  1. A comparative study on the aggregating effects of guanidine thiocyanate, guanidine hydrochloride and urea on lysozyme aggregation

    Energy Technology Data Exchange (ETDEWEB)

    Emadi, Saeed, E-mail:; Behzadi, Maliheh


    Highlights: • Lysozyme aggregated in guanidine thiocyanate (1.0 and 2.0 M). • Lysozyme aggregated in guanidine hydrochloride (4 and 5 M). • Lysozyme did not aggregated at any concentration (0.5–5 M) of urea. • Unfolding pathway is more important than unfolding per se in aggregation. - Abstract: Protein aggregation and its subsequent deposition in different tissues culminate in a diverse range of diseases collectively known as amyloidoses. Aggregation of hen or human lysozyme depends on certain conditions, namely acidic pH or the presence of additives. In the present study, the effects on the aggregation of hen egg-white lysozyme via incubation in concentrated solutions of three different chaotropic agents namely guanidine thiocyanate, guanidine hydrochloride and urea were investigated. Here we used three different methods for the detection of the aggregates, thioflavin T fluorescence, circular dichroism spectroscopy and atomic force microscopy. Our results showed that upon incubation with different concentrations (0.5, 1.0, 2.0, 3.0, 4.0, 5.0 M) of the chemical denaturants, lysozyme was aggregated at low concentrations of guanidine thiocyanate (1.0 and 2.0 M) and at high concentrations of guanidine hydrochloride (4 and 5 M), although no fibril formation was detected. In the case of urea, no aggregation was observed at any concentration.

  2. Time-dependent X-ray diffraction studies on urea/hen egg white lysozyme complexes reveal structural changes that indicate onset of denaturation (United States)

    Raskar, Tushar; Khavnekar, Sagar; Hosur, Madhusoodan


    Temporal binding of urea to lysozyme was examined using X-ray diffraction of single crystals of urea/lysozyme complexes prepared by soaking native lysozyme crystals in solutions containing 9 M urea. Four different soak times of 2, 4, 7 and 10 hours were used. The five crystal structures (including the native lysozyme), refined to 1.6 Å resolution, reveal that as the soaking time increased, more and more first-shell water molecules are replaced by urea. The number of hydrogen bonds between urea and the protein is similar to that between protein and water molecules replaced by urea. However, the number of van der Waals contacts to protein from urea is almost double that between the protein and the replaced water. The hydrogen bonding and van der Waals interactions are initially greater with the backbone and later with side chains of charged residues. Urea altered the water-water hydrogen bond network both by replacing water solvating hydrophobic residues and by shortening the first-shell intra-water hydrogen bonds by 0.2 Å. These interaction data suggest that urea uses both ‘direct’ and ‘indirect’ mechanisms to unfold lysozyme. Specific structural changes constitute the first steps in lysozyme unfolding by urea. PMID:27573790

  3. Nonequivalence of second virial coefficients from sedimentation equilibrium and static light scattering studies of protein solutions. (United States)

    Winzor, Donald J; Deszczynski, Marcin; Harding, Stephen E; Wills, Peter R


    Experimental data for ovalbumin and lysozyme are presented to highlight the nonequivalence of second virial coefficients obtained for proteins by sedimentation equilibrium and light scattering. Theoretical considerations confirm that the quantity deduced from sedimentation equilibrium distributions is B(22), the osmotic second virial coefficient describing thermodynamic nonideality arising solely from protein self-interaction. On the other hand, the virial coefficient determined by light scattering is shown to reflect the combined contributions of protein-protein and protein-buffer interactions to thermodynamic nonideality of the protein solution. Misidentification of the light scattering parameter as B(22) accounts for published reports of negative osmotic second virial coefficients as indicators of conditions conducive to protein crystal growth. Finally, textbook assertions about the equivalence of second virial coefficients obtained by sedimentation equilibrium and light scattering reflect the restriction of consideration to single-solute systems. Although sedimentation equilibrium distributions for buffered protein solutions are, indeed, amenable to interpretation in such terms, the same situation does not apply to light scattering measurements because buffer constituents cannot be regarded as part of the solvent: instead they must be treated as non-scattering cosolutes.

  4. Complex coacervates of hyaluronic acid and lysozyme

    DEFF Research Database (Denmark)

    Water, Jorrit J.; Schack, Malthe M.; Velazquez-Campoy, Adrian;


    Complex coacervates of hyaluronic acid and lysozyme, a model protein, were formed by ionic interaction using bulk mixing and were characterized in terms of binding stoichiometry and protein structure and stability. The complexes were formed at pH 7.2 at low ionic strength (6 mM) and the binding s...

  5. Yam tuber mucilage as a candidate substance for saliva substitute: in vitro study of its viscosity and influences on lysozyme and peroxidase activities. (United States)

    Kho, Hong-Seop; Park, Moon-Soo; Chang, Ji-Youn; Kim, Yoon-Young


    To investigate the viscosity of yam tuber mucilage (YTM) and its effects on lysozyme and peroxidase activities in solution phase and on surface phase. Two kinds of YTM were extracted, one containing both protein and carbohydrate and the other containing mainly carbohydrate. Hen egg-white lysozyme and bovine lactoperoxidase were used as lysozyme and peroxidase sources, respectively. Viscosity was measured with a cone-and-plate digital viscometer. Lysozyme activity was determined using the turbidimetric method, and peroxidase activity was determined using the NbsSCN assay. Hydroxyapatite beads were used as a solid phase. The viscosity values of YTM followed a pattern of a non-Newtonian fluid. The carbohydrate concentration affected the viscosity values at all shear rates, while the protein concentration affected the viscosity values at low shear rates. It could be suggested that YTM composed of 1.0 mg/ml protein and 1.0 mg/ml carbohydrate has viscosity values similar to those of unstimulated whole saliva at shear rates present at routine oral functions. Hydroxyapatite-adsorbed YTM significantly increased the adsorption and subsequent enzymatic activities of lysozyme, but not those of peroxidase. Yam tuber mucilage has viscoelastic properties similar to those of human saliva and enhances the enzymatic activity of lysozyme on hydroxyapatite surfaces. © 2012 John Wiley & Sons A/S and The Gerodontology Association. Published by John Wiley & Sons Ltd.


    Directory of Open Access Journals (Sweden)

    S. S. Dekina


    Full Text Available The lysozyme immobilization in cryogel of polyvinyl alcohol and physico-chemical properties of obtained preparation was investigated. Hydrolytic activity of lysozyme was determined by bacteriolytic method, using Micrococcus lysodeikticus cells acetone powder as substrate. Protein content was determined by the Lowry–Hartree method. Immobilization of lysozyme was conducted by entrapment in polyvinyl alcohol gel with subsequent cycles of freezing-thawing. Antimicrobial activity was studied by standard disk-diffusional method. The hydrogel filmic coatings with antimicrobial action, insoluble at physiological conditions, with quantitative retaining of protein and hydrolytic activity of lysozyme were obtained. The product is characterized by the widened pH-profile of activity at acidic pH values, stability in acidic medium (pH 5.5 and at storage. Its antimicrobial action against Staphylococcus aureus ATCC 25923 F-49, Pseudomonas aeruginosa 415, Escherichia coli 055 K 59912/4 and Candida albicans ATCC 885-653 was noted. The proposed method of lysozyme immobilization allows to obtain stable, highly effective product with antimicrobial activity, prospective for usage in biomedical investigations.

  7. Synthesis of Novel Phosphorylated Daidzein Derivatives and ESI Investigation on Their Non-Covalent Complexes with Lysozyme

    Institute of Scientific and Technical Information of China (English)

    CHEN, Xiao-Lan; SHI, Xiao-Na; QU, Ling-Bo; YUAN, Jin-Wei; LU, Jian-Sha; ZHAO, Yu-Fen


    Daidzein (7,4'-dihydroxyisoflavone) was phosphorylated by a modified Atherton-Todd reaction. The structures of the five target product, were determined by X-ray, IR, NMR and ESI-MS. Electrospray ionization results show that in the gas phase all the phosphorylated daidzein derivatives could form non-covalent complexes with the protein lysozyme, while non-covalent complexes were not detected in the mixed solution of daidzein with lysozyme.Relative affinity of every non-covalent complex was obtained according to its different decomposition orifice voltage.

  8. Solution blowing of soy protein fibers. (United States)

    Sinha-Ray, S; Zhang, Y; Yarin, A L; Davis, S C; Pourdeyhimi, B


    Solution blowing of soy protein (sp)/polymer blends was used to form monolithic nanofibers. The monolithic fibers were blown from blends of soy protein and nylon-6 in formic acid. The sp/nylon-6 ratio achieved in dry monolithic nanofibers formed using solution blowing of the blend was equal to 40/60. In addition, solution blowing of core-shell nanofibers was realized with soy protein being in the core and the supporting polymer in the shell. The shells were formed from nylon-6. The sp/nylon-6 ratio achieved in dry core-shell fibers was 32/68. The nanofibers developed in the present work contain significant amounts of soy protein and hold great potential in various applications of nonwovens.

  9. Aptamer-Based Electrochemical Sensing of Lysozyme

    Directory of Open Access Journals (Sweden)

    Alina Vasilescu


    Full Text Available Protein analysis and quantification are required daily by thousands of laboratories worldwide for activities ranging from protein characterization to clinical diagnostics. Multiple factors have to be considered when selecting the best detection and quantification assay, including the amount of protein available, its concentration, the presence of interfering molecules, as well as costs and rapidity. This is also the case for lysozyme, a 14.3-kDa protein ubiquitously present in many organisms, that has been identified with a variety of functions: antibacterial activity, a biomarker of several serious medical conditions, a potential allergen in foods or a model of amyloid-type protein aggregation. Since the design of the first lysozyme aptamer in 2001, lysozyme became one of the most intensively-investigated biological target analytes for the design of novel biosensing concepts, particularly with regards to electrochemical aptasensors. In this review, we discuss the state of the art of aptamer-based electrochemical sensing of lysozyme, with emphasis on sensing in serum and real samples.

  10. Mechanic Insight into Aggregation of Lysozyme by Ultrasensitive Differential Scanning Calorimetry and Sedimentation Velocity. (United States)

    Wu, Sha; Ding, Yanwei; Zhang, Guangzhao


    Folding and aggregation of proteins profoundly influence their functions. We have investigated the effects of thermal history, concentration and pH on the denaturation and refolding of lysozyme by using ultrasensitive differential scanning calorimetry (US-DSC) and sedimentation velocity (SV) via analytical ultracentrifugation (AUC). The former is sensitive to small energy change whereas the latter can differentiate the oligomers such as dimer and trimer from individual protein molecules. Our studies reveal that the degree of denaturation irreversibility increases as heating times increases. The denaturation temperature (Td) and enthalpy change (ΔH) are influenced by heating rate since the denaturation is not in equilibrium during the heating. We can obtain Td and ΔH in equilibrium by extrapolation of heating rate to zero. In a dilute solution, no aggregation but unfolding happens in the denaturation. However, when the concentration is above a critical value (∼15.0 mg/mL), lysozyme molecules readily form trimers or other oligomers. Lysozyme molecules unfold into stretched chains at pH > 6.0, which would further forms large aggregates. The formation of aggregates makes the refolding of lysozyme impossible.

  11. Crystal Growth of Hen Egg-White Lysozyme (HEWL) under Various Gravity Conditions (United States)

    Pan, Weichun; Xu, Jin; Tsukamoto, Katsuo; Koizumi, Masako; Yamazaki, Tomoya; Zhou, Ru; Li, Ang; Fu, Yuying


    Motivated by the enhancement of protein quality under microgravity condition, the behaviors of crystal growth under various gravity conditions have been monitored via Foton Satellite and parabolic flight. We found that the normal growth rate and the step velocity would be enhanced only at high protein concentration. Although the difference of diffusion between monomer lysozyme molecule and main impurity species in HWEL dimer may be able to explain this enhancement in long period at high protein concentration, it is not valid at low lysozyme concentration and it can't explain the results obtained by parabolic flight, in which microgravity condition maintained only about 20 s. In order to compromise this contradiction, cluster, universal existing in protein solution, has been picked up. The dynamic light scattering technique figured out dimer is served as the seed for cluster formation. Due to its large size, cluster keeps still under microgravity. Via this mechanism, the purification of lysozyme above crystal surface has been achieved. We also found the two supergravity (˜1.5 g) periods immediately before and after microgravity period have different effects on the step velocity. The pre-MG period depresses the step velocity while the post-MG enhances it. This odd phenomenon ascribes to two factors: (1) the flow rate modification and (2) the purity of protein solution immediate above crystal surface.

  12. Optimization of buffer solutions for protein crystallization. (United States)

    Gosavi, Rajendrakumar A; Mueser, Timothy C; Schall, Constance A


    Increasing the solubility of protein stock solutions to above that in a standard chromatography buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl) led to an increase in the number of crystallization conditions for ten globular proteins subjected to two crystal screens: the Index and Precipitant/Precipitant-Additive (P/PA) Screens. Solubility enhancement of protein stock solutions was achieved through screening and selection of buffer components to formulate an optimal buffer. Relative improvements in solubility were estimated through protection against the precipitation of protein by polyethylene glycol 8000. Proteins with limited solubility improvement in optimal buffer showed an enhancement in solubility on addition of glycerol. Maximum solubility was then determined by the concentration of optimized solutions until precipitate formed. The supernatant concentration then provided an estimate of the upper limit of protein solubility. This 'solubility' estimate is used to specify the initial concentration of the protein used in the screening experiments and is an important step in successful crystallization. Buffer optimization and establishment of initial protein concentration for crystal screening based on solubility estimates provides a methodology for improved crystal screening results.

  13. Nucleation of protein crystals under the influence of solution shear flow. (United States)

    Penkova, Anita; Pan, Weichun; Hodjaoglu, Feyzim; Vekilov, Peter G


    Several recent theories and simulations have predicted that shear flow could enhance, or, conversely, suppress the nucleation of crystals from solution. Such modulations would offer a pathway for nucleation control and provide a novel explanation for numerous mysteries in nucleation research. For experimental tests of the effects of shear flow on protein crystal nucleation, we found that if a protein solution droplet of approximately 5 microL (2-3 mm diameter at base) is held on a hydrophobic substrate in an enclosed environment and in a quasi-uniform constant electric field of 2 to 6 kV cm(-1), a rotational flow with a maximum rate at the droplet top of approximately 10 microm s(-1) is induced. The shear rate varies from 10(-3) to 10(-1) s(-1). The likely mechanism of the rotational flow involves adsorption of the protein and amphiphylic buffer molecules on the air-water interface and their redistribution in the electric field, leading to nonuniform surface tension of the droplet and surface tension-driven flow. Observations of the number of nucleated crystals in 24- and 72-h experiments with the proteins ferritin, apoferritin, and lysozyme revealed that the crystals are typically nucleated at a certain radius of the droplet, that is, at a preferred shear rate. Variations of the rotational flow velocity resulted in suppression or enhancement of the total number of nucleated crystals of ferritin and apoferritin, while all solution flow rates were found to enhance lysozyme crystal nucleation. These observations show that shear flow may strongly affect nucleation, and that for some systems, an optimal flow velocity, leading to fastest nucleation, exists. Comparison with the predictions of theories and simulations suggest that the formation of ordered nuclei in a "normal" protein solution cannot be affected by such low shear rates. We conclude that the flow acts by helping or suppressing the formation of ordered nuclei within mesoscopic metastable dense liquid

  14. Lysozyme Aggregation and Fibrillation Monitored by Dynamic Light Scattering (United States)

    Nemzer, Louis; Flanders, Bret; Schmit, Jeremy; Sorensen, Christopher


    The aggregation of amyloidogenic proteins provides a rich phase space with significant biomedical implications, including a link with several age-related diseases. We employed dynamic light scattering to monitor the aggregation of lysozyme, a model protein, from a monomeric state until the formation of micron-sized fibrils. For an aqueous lysozyme solution buffered at pH 2, the auto-correlation function of the scattered light intensity was found to be well-fit by a single exponential function with decay time τ = 1/(2Dq^2) = 0.25 ms, which corresponds to a mean hydrodynamic radius (RH) of 2.2 nm, very likely generated by monomers. Ethanol (4% v/v final concentration) induced a partial unfolding, to RH = 4.6 nm. The subsequent addition of 70 mM KCl was found to shrink the size back to RH = 2.5 nm, as expected when a denatured protein refolds due to partial screening of the intramolecular repulsion. However, further aggregation was not observed. At pH 4, using a low-salt acetate buffer, more ethanol (10% v/v) was required to initiate unfolding, but once it occurred, larger aggregates formed. These results are consistent with the model that partial unfolding, which exposes beta-motif secondary structure, is a prerequisite for aggregation and fibrillation, but the aggregation fate depends on the protein charge state (pH) and screening (salt concentration).

  15. Viscosity measurements of antibody solutions by photon correlation spectroscopy: an indirect approach - limitations and applicability for high-concentration liquid protein solutions. (United States)

    Wagner, Michael; Reiche, Katharina; Blume, Alfred; Garidel, Patrick


    Photon correlation spectroscopy (PCS) is compared with classic rheological measurements using the cone-and-plate technique for the determination of the viscosity of protein solutions. The potential advantages using PCS are small sample volume and fast determination of zero-shear viscosity. The present study assesses potentials and limitations of the applicability of this method for the determination of viscosity of antibody solutions in protein science development. The principle of the assay is based on the determination of the apparent hydrodynamic radius of commercial available latex beads of known size added to protein solutions. Using the Stokes-Einstein equation, the hydrodynamic radius can be converted to viscosity. Several latex particle sizes and concentrations were evaluated and the assay optimized. The PCS assay for viscosity determination was tested using water/glycerol-mixtures, where the viscosity was measured with rheometer using the cone-and-plate method and also compared with published data. Different protein solutions of bovine serum albumin, lysozyme and monoclonal antibodies were then used and the PCS results were compared with viscosity data obtained by the cone-and-plate method. It could be shown that the PCS assay has limitations for the determination of the viscosity of protein solutions, especially monoclonal antibodies. The main reason is due to protein-latex bead interactions leading to the formation of larger aggregates. The use of surface modification of the latex beads can in principle prevent this interaction.

  16. Lysozyme uptake by oxidized starch polymer microgels

    NARCIS (Netherlands)

    Li, Y.; Vries, R.D.; Kleijn, M.; Slaghek, T.; Timmermans, J.; Stuart, M.C.; Norde, W.


    With the aim of determining suitable conditions for uptake and release of globular proteins on microgels, we studied the interaction between phosphated, highly cross-linked, negatively charged oxidized potato starch polymer (OPSP) microgel particles and lysozyme from hen eggs. Our microgel shows a

  17. Lysozyme expression in Lactococcus lactis

    NARCIS (Netherlands)

    Guchte, Maarten van de; Wal, Fimme Jan van der; Kok, Jan; Venema, Gerhardus


    Three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in Lactococcus lactis subsp. lactis. Hen egg white lysozyme (HEL) could be detected in L. lactis lysates by Western blotting. No lysozyme activity was observed, however, presumably because of the absence o

  18. Can Solution Supersaturation Affect Protein Crystal Quality? (United States)

    Gorti, Sridhar


    The formation of large protein crystals of "high quality" is considered a characteristic manifestation of microgravity. The physical processes that predict the formation of large, high quality protein crystals in the microgravity environment of space are considered rooted in the existence of a "depletion zone" in the vicinity of crystal. Namely, it is considered reasonable that crystal quality suffers in earth-grown crystals as a result of the incorporation of large aggregates, micro-crystals and/or large molecular weight "impurities", processes which are aided by density driven convective flow or mixing at the crystal-liquid interface. Sedimentation and density driven convection produce unfavorable solution conditions in the vicinity of the crystal surface, which promotes rapid crystal growth to the detriment of crystal size and quality. In this effort, we shall further present the hypothesis that the solution supersaturatoin at the crystal surface determines the growth mechanism, or mode, by which protein crystals grow. It is further hypothesized that protein crystal quality is affected by the mechanism or mode of crystal growth. Hence the formation of a depletion zone in microgravity environment is beneficial due to inhibition of impurity incorporatoin as well as preventing a kinetic roughening transition. It should be noted that for many proteins the magnitude of neither protein crystal growth rates nor solution supersaturation are predictors of a kinetic roughening transition. That is, the kinetic roughening transition supersaturation must be dtermined for each individual protein.

  19. Influence of precipitating agents on thermodynamic parameters of protein crystallization solutions. (United States)

    Stavros, Philemon; Saridakis, Emmanuel; Nounesis, George


    X-ray crystallography is the most powerful method for determining three-dimensional structures of proteins to (near-)atomic resolution, but protein crystallization is a poorly explained and often intractable phenomenon. Differential Scanning Calorimetry was used to measure the thermodynamic parameters (ΔG, ΔH, ΔS) of temperature-driven unfolding of two globular proteins, lysozyme, and ribonuclease A, in various salt solutions. The mixtures were categorized into those that were conducive to crystallization of the protein and those that were not. It was found that even fairly low salt concentrations had very large effects on thermodynamic parameters. High concentrations of salts conducive to crystallization stabilized the native folded forms of proteins, whereas high concentrations of salts that did not crystallize them tended to destabilize them. Considering the ΔH and TΔS contributions to the ΔG of unfolding separately, high concentrations of crystallizing salts were found to enthalpically stabilize and entropically destabilize the protein, and vice-versa for the noncrystallizing salts. These observations suggest an explanation, in terms of protein stability and entropy of hydration, of why some salts are good crystallization agents for a given protein and others are not. This in turn provides theoretical insight into the process of protein crystallization, suggesting ways of predicting and controlling it. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 642-652, 2016.

  20. Adaptive functional diversification of lysozyme in insectivorous bats. (United States)

    Liu, Yang; He, Guimei; Xu, Huihui; Han, Xiuqun; Jones, Gareth; Rossiter, Stephen J; Zhang, Shuyi


    The role of gene duplication in generating new genes and novel functions is well recognized and is exemplified by the digestion-related protein lysozyme. In ruminants, duplicated chicken-type lysozymes facilitate the degradation of symbiotic bacteria in the foregut. Chicken-type lysozyme has also been reported to show chitinase-like activity, yet no study has examined the molecular evolution of lysozymes in species that specialize on eating insects. Insectivorous bats number over 900 species, and lysozyme expression in the mouths of some of these species is associated with the ingestion of insect cuticle, suggesting a chitinase role. Here, we show that chicken-type lysozyme has undergone multiple duplication events in a major family of insect-eating bats (Vespertilionidae) and that new duplicates have undergone molecular adaptation. Examination of duplicates from two insectivorous bats-Pipistrellus abramus and Scotophilus kuhlii-indicated that the new copy was highly expressed in the tongue, whereas the other one was less tissue-specific. Functional assays applied to pipistrelle lysozymes confirmed that, of the two copies, the tongue duplicate was more efficient at breaking down glycol chitin, a chitin derivative. These results suggest that the evolution of lysozymes in vespertilionid bats has likely been driven in part by natural selection for insectivory.

  1. Refolding of Denatured/Reduced Lysozyme Using Weak-Cation Exchange Chromatography

    Institute of Scientific and Technical Information of China (English)

    Yan WANG; Bo Lin GONG; Xin Du GENG


    Oxidative refolding of the denatured/reduced lysozyme was investigated by using weak-cation exchange chromatography (WCX). The stationary phase of WCX binds to the reduced lysozyme and prevented it from forming intermolecular aggregates. At the same time urea and ammonium sulfate were added to the mobile phase to increase the elution strength for lysozyme. Ammonium sulfate can more stabilize the native protein than a common eluting agent, sodium chloride. Refolding of lysozyme by using this WCX is successfully. It was simply carried out to obtain a completely and correctly refolding of the denatured lysozyme at high concentration of 20.0 mg/mL.

  2. Thermodynamic Properties of Linear Protein Solutions: an Application to Type Ⅰ Antifreeze Protein Solutions

    Institute of Scientific and Technical Information of China (English)

    LI Li-fen; LIANG Xi-xia; LI Qian-zhong


    A statistical thermodynamic theory of linear protein solutions was proposed with the aid of a lattice model and applied to type Ⅰ antifreeze protein(AFPI) solutions.The numerical results for several AFPI solutions show that the Gibbs function of the solution has a minimum at a certain protein concentration,but the protein chemical potential increases with increasing the concentration.The influences of temperature and protein chain length on the AFPI chemical potential were also discussed.The evaluation for the colligative depression of the freezing point confirms that the antifreeze action should be recognized as non-colligative.The theoretical deduction for the concentration dependence of the thermal hysteresis activity coincides qualitatively with the previous experimental and theoretical results.

  3. Introduction of Ca(2+)-binding amino-acid sequence into the T4 lysozyme. (United States)

    Leontiev, V V; Uversky, V N; Permyakov, E A; Murzin, A G


    The 51-62 loop of T4 phage lysozyme was altered by site-directed mutagenesis to obtain maximal homology with the typical EF-hand motif. A Ca(2+)-binding site was designed and created by replacing both Gly-51 and Asn-53 with aspartic acid. The mutant T4 lysozyme (G51D/N53D) was expressed in Escherichia coli. The activity of the G51D/N53D-mutant was about 60% of that of the wild-type protein. This mutant can bind Ca2+ ions specifically, while the effective dissociation constant was essentially greater than that of the EF-hand proteins. Stability of the G51D/N53D-mutant apo-form to urea- or temperature-induced denaturation was the same as that of the wild-type protein. In the presence of Ca2+ ions in solution the stability of the mutant T4 phage lysozyme was less than that of the wild-type protein. It is suggested that the binding of Ca2+ by the mutant is accompanied by the considerable conformational changes in the 'corrected' loop, which can lead to the Ca(2+)-induced destabilization of the protein.

  4. Lysozymes in the animal kingdom

    Indian Academy of Sciences (India)

    Lien Callewaert; Chris W Michiels


    Lysozymes (EC are hydrolytic enzymes, characterized by their ability to cleave the -(1,4)-glycosidic bond between -acetylmuramic acid and -acetylglucosamine in peptidoglycan, the major bacterial cell wall polymer. In the animal kingdom, three major distinct lysozyme types have been identified – the c-type (chicken or conventional type), the g-type (goose-type) and the i-type (invertebrate type) lysozyme. Examination of the phylogenetic distribution of these lysozymes reveals that c-type lysozymes are predominantly present in the phylum of the Chordata and in different classes of the Arthropoda. Moreover, g-type lysozymes (or at least their corresponding genes) are found in members of the Chordata, as well as in some bivalve mollusks belonging to the invertebrates. In general, the latter animals are known to produce i-type lysozymes. Although the homology in primary structure for representatives of these three lysozyme types is limited, their three-dimensional structures show striking similarities. Nevertheless, some variation exists in their catalytic mechanisms and the genomic organization of their genes. Regarding their biological role, the widely recognized function of lysozymes is their contribution to antibacterial defence but, additionally, some lysozymes (belonging to different types) are known to function as digestive enzymes.

  5. Preparation of lysozyme molecularly imprinted polymers and purification of lysozyme from egg white. (United States)

    Wang, Xuejiao; Dong, Shaohua; Bai, Quan


    Molecular imprinting as a promising and facile separation technique has received much attention because of its high selectivity for target molecules. In this study, lysozyme molecularly imprinted polymers (Lys-MIPs) were successfully prepared by the entrapment method with lysozyme as the template molecule, acrylamide as the functional monomer and N,N-methylenebisacrylamide as the cross-linker. The removal of the template lysozyme from the molecularly imprinted polymers was investigated in detail by two methods. The synthesized Lys-MIPs were characterized by scanning electron microscopy and Fourier transform-infrared, and the adsorption capacity, selectivity and reproducibility of the Lys-MIPs were also evaluated. The maximum adsorption capacity reached 94.8 mg/g, which is twice that of nonmolecularly imprinted polymers, and satisfactory selectivity and reproducibility were achieved. Using the Lys-MIP column, lysozyme could be separated completely from egg white, with purity close to 100% and mass recovery of 98.2%. This illustrated that the synthesized Lys-MIPs had high specific recognition and selectivity to the template lysozyme when they were applied to a mixture of protein standards and a real sample.

  6. Investigation of protein distribution in solid lipid particles and its impact on protein release using coherent anti-Stokes Raman scattering microscopy

    DEFF Research Database (Denmark)

    Christophersen, Philip C.; Birch, Ditlev; Saarinen, Jukka


    The aim of this study was to gain new insights into protein distribution in solid lipid microparticles (SLMs) and subsequent release mechanisms using a novel label-free chemical imaging method, coherent anti-Stokes Raman scattering (CARS) microscopy. Lysozyme-loaded SLMs were prepared using...... different lipids with lysozyme incorporated either as an aqueous solution or as a solid powder. Lysozyme distribution in SLMs was investigated using CARS microscopy with supportive structural analysis using electron microscopy. The release of lysozyme from SLMs was investigated in a medium simulating......-destructive method for elucidating the distribution of lysozyme in SLMs. The interpretation of protein distribution and release during lipolysis enabled elucidation of protein release mechanisms. In future, CARS microscopy analysis could facilitate development of a wide range of protein-lipid matrices with tailor...


    Institute of Scientific and Technical Information of China (English)

    Hong-wei Wang; Lin Yuan; Tie-liang Zhao; He Huang; Hong Chen; Di Wu


    The purpose of this research is to investigate the effects of the variously sulfated chitosans on lysozyme activity and structure.It was shown that the specific enzymatic activity of lysozyme remained almost similar to the native protein after being bound to 6-O-sulfated chitosan (6S-chitosan) and 3,6-O-sulfated chitosan (3,6S-chitosan),but decreased greatly after being bound to 2-N-6-O-sulfated chitosan (2,6S-chitosan).Meanwhile,among these sulfated chitosans,2,6S-chitosan induced the greatest conformational change in lysozyme as indicated by the fluorescence spectra.These findings demonstrated that when sulfated chitosans of different structures bind to lysozyme,lysozyme undergoes conformational change of different magnitudes,which results in corresponding levels of lysozyme activity.Further study on the interaction of sulfated chitosans with lysozyme by surface plasmon resonance (SPR) suggested that their affinities might be determined by their molecular structures.

  8. Lysozyme Catalyzes the Formation of Antimicrobial Silver Nanoparticles (POSTPRINT) (United States)


    aseptics and therapeutic use in the future. KEYWORDS: antimicrobial · lysozyme · silver · nanoparticle · biocompatibility · biomineralization A RT IC LE VOL...protein that will adsorb to ionic and hydro- phobic surfaces, including metal surfaces.2124 After synthesis in methanol, lysozyme-stabilized nanoparti- cle...the strong ionic interactions be- tween metal nanoparticles normally make it difficult to achieve high concentrations of monodispersed and stable

  9. Three Frontiers in the Thermodynamics of Protein Solutions

    Energy Technology Data Exchange (ETDEWEB)

    Prausnitz, John; Hagar, Loddie


    Three examples illustrate the versatility and usefulness of biothermodynamics. The first example concerns calculation of a phase diagram for aqueous lysozyme with a new potential of mean force that takes the Hofmeister effect into account; such calculations may be useful for design of a separation process where addition of a salt to an aqueous protein mixture precipitates a target protein. The second example concerns thermodynamic studies to elucidate the effect of an organic cosolvent on the mechanism of crystallizing aqueous insulin. The final example concerns a thermodynamic contribution to mitigating the AIDS epidemic; it indicates how isothermal-titration-calorimetry studies are helpful for choosing an optimum inhibitor that is effective not only for the wild-type HIV protease but also for at least some of its mutants.

  10. Studies of protein structure in solution and protein folding using synchrotron small-angle x-ray scattering

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Lingling [Stanford Univ., CA (United States)


    Synchrotron small angle x-ray scattering (SAXS) has been applied to the structural study of several biological systems, including the nitrogenase complex, the heat shock cognate protein (hsc70), and lysozyme folding. The structural information revealed from the SAXS experiments is complementary to information obtained by other physical and biochemical methods, and adds to our knowledge and understanding of these systems.

  11. A comparative study for adsorption of lysozyme from aqueous samples onto Fe3O4 magnetic nanoparticles using different ionic liquids as modifier. (United States)

    Kamran, Sedigheh; Absalan, Ghodratollah; Asadi, Mozaffar


    In this paper, nanoparticles of Fe3O4 as well as their modified forms with different ionic liquids (IL-Fe3O4) were prepared and used for adsorption of lysozyme. The mean size and the surface morphology of the nanoparticles were characterized by TEM, XRD and FTIR techniques. Adsorption studies of lysozyme were performed under different experimental conditions in batch system on different modified magnetic nanoparticles such as, lysozyme concentration, pH of the solution, and contact time. Experimental results were obtained under the optimum operational conditions of pH 9.0 and a contact time of 10 min when initial protein concentrations of 0.05-2.0 mg mL(-1) were used. The isotherm evaluations revealed that the Langmuir model attained better fits to the equilibrium data than the Freundlich model. The maximum obtained adsorption capacities were 370.4, 400.0 500.0 and 526.3 mg of lysozyme for adsorption onto Fe3O4 and modified magnetic nanoparticles by [C4MIM][Br], [C6MIM][Br] and [C8MIM][Br] per gram of adsorbent, respectively. The Langmuir adsorption constants were 0.004, 0.019, 0.024 and 0.012 L mg(-1) for adsorptions of lysozyme onto Fe3O4 and modified magnetic nanoparticles by [C4MIM][Br], [C6MIM][Br] and [C8MIM][Br], respectively. The adsorption capacity of lysozyme was found to be dependent on its chemical structure, pH of the solution, temperature and type of ionic liquid as modifier. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated. Furthermore, the thermodynamic parameters were calculated. Protein could desorb from IL-Fe3O4 nanoparticles by using NaCl solution at pH 9.5 and was reused.

  12. Toward optimized potential functions for protein-protein interactions in aqueous solutions: osmotic second virial coefficient calculations using the MARTINI coarse-grained force field. (United States)

    Stark, Austin C; Andrews, Casey T; Elcock, Adrian H


    Coarse-grained (CG) simulation methods are now widely used to model the structure and dynamics of large biomolecular systems. One important issue for using such methods - especially with regard to using them to model, for example, intracellular environments - is to demonstrate that they can reproduce experimental data on the thermodynamics of protein-protein interactions in aqueous solutions. To examine this issue, we describe here simulations performed using the popular coarse-grained MARTINI force field, aimed at computing the thermodynamics of lysozyme and chymotrypsinogen self-interactions in aqueous solution. Using molecular dynamics simulations to compute potentials of mean force between a pair of protein molecules, we show that the original parameterization of the MARTINI force field is likely to significantly overestimate the strength of protein-protein interactions to the extent that the computed osmotic second virial coefficients are orders of magnitude more negative than experimental estimates. We then show that a simple down-scaling of the van der Waals parameters that describe the interactions between protein pseudo-atoms can bring the simulated thermodynamics into much closer agreement with experiment. Overall, the work shows that it is feasible to test explicit-solvent CG force fields directly against thermodynamic data for proteins in aqueous solutions, and highlights the potential usefulness of osmotic second virial coefficient measurements for fully parameterizing such force fields.

  13. Destroying activity of magnetoferritin on lysozyme amyloid fibrils

    Energy Technology Data Exchange (ETDEWEB)

    Kopcansky, Peter; Siposova, Katarina [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Melnikova, Lucia, E-mail: [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Bednarikova, Zuzana [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Institute of Chemical Sciences, Faculty of Sciences, Safarik University, Kosice (Slovakia); Timko, Milan; Mitroova, Zuzana; Antosova, Andrea [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Garamus, Vasil M. [Helmholtz-Zentrum Geesthacht: Centre for Materials and Coastal Research, Max-Planck-Street 1, 21502 Geesthacht (Germany); Petrenko, Viktor I. [Joint Institute for Nuclear Research, Joliot-Curie 6, Dubna, 141980 Moscow Region (Russian Federation); Kyiv Taras Shevchenko National University, Volodymyrska Street 64, Kyiv 01033 (Ukraine); Avdeev, Mikhail V. [Joint Institute for Nuclear Research, Joliot-Curie 6, Dubna, 141980 Moscow Region (Russian Federation); Gazova, Zuzana [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Department of Medical and Clinical Biochemistry and LABMED, Tr. SNP 1, 040 11 Kosice (Slovakia)


    Presence of protein amyloid aggregates (oligomers, protofilaments, fibrils) is associated with many diseases as diabetes mellitus or Alzheimer's disease. The interaction between lysozyme amyloid fibrils and magnetoferritin loaded with different amount of iron atoms (168 or 532 atoms) has been investigated by small-angle X-rays scattering and thioflavin T fluorescence measurements. Results suggest that magnetoferritin caused an iron atom-concentration dependent reduction of lysozyme fibril size. - Highlights: • The interaction between lysozyme amyloid fibrils and magnetoferritin loaded with different amount of iron atoms (168 or 532 atoms) has been investigated by small-angle X-rays scattering and thioflavin T fluorescence measurements. • Results suggest that magnetoferritin caused an iron atom-concentration dependent reduction of lysozyme fibril size.

  14. Lysozyme complexes with thermo- and pH-responsive PNIPAM- b-PAA block copolymer (United States)

    Pippa, Natassa; Meristoudi, Anastasia; Pispas, Stergios; Demetzos, Costas


    Lysozyme is an enzyme responsible for the damage of bacterial cell walls and is abundant in a number of secretions such as tears and human milk. In the present study, we investigated the structure, the physicochemical characteristics, and the temperature-responsiveness of lysozyme complexes with poly( N-isopropylacrylamide)- b-poly(acrylic acid) block polyelectrolyte in aqueous media. A gamut of light-scattering techniques and fluorescence spectroscopy were used in order to examine the complexation process, as well as the structure, solution behavior, and temperature response of the nanosized complexes. The concentration of copolymer polyelectrolyte was kept constant. The values of the scattering intensity, I 90, which is proportional to the mass of the species in solution, increased gradually as a function of C LYS, providing proof of the occurring complexation, while the size of the nanostructures decreased. The structure of the complexes became more open as the C LYS increased. The increase of the salinity did not affect the structural characteristics of the supramolecular nanoparticulate aggregates. On the other hand, the physicochemical and structural characteristics of the complexes changed upon increasing temperature, and the changes depended on the initial ratio block polyelectrolyte/lysozyme. The knowledge on developing block polyelectrolyte/protein complexes through electrostatic interactions, obtained from this investigation, may be applied to the design of nutraceuticals.

  15. Lysozyme Photochemistry as a Function of Temperature. The Protective Effect of Nanoparticles on Lysozyme Photostability (United States)

    Oliveira Silva, Catarina; Petersen, Steffen B.; Pinto Reis, Catarina; Rijo, Patrícia; Molpeceres, Jesús; Vorum, Henrik; Neves-Petersen, Maria Teresa


    The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N–formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 μW and 0.3 μW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a

  16. Hydrogen-ion titrations of amino acids and proteins in solutions containing concentrated electrolyte

    Energy Technology Data Exchange (ETDEWEB)

    Fergg, F. [Technische Universitaet Muenchen (Germany); Kuehner, D.E.; Blanch, H.W.; Prausnitz, J.M. [Lawrence Berkeley Lab., CA (United States)


    This report describes a first attempt to quantify the net charge as a function of solution pH for lysozyme and {alpha}-chymotrypsin at 0.1 M, 1.0 M and 3.0 M ionic strength, (IS). The calculations are based on the residue (titratable group) pK{sub a}`s in the amino-acid sequence of the protein. To determine these pK{sub a}`s, a simple theory was used which assumes that the pK{sub a}`s are independent from each other in the protein and are equal to their pK{sub a} values in free amino-acid solution (Independent-Site Theory, IST). Residue pK{sub a}`s were obtained from amino-acid hydrogen-ion titrations at three different KCl concentrations corresponding to 0.1M, 1.0M and 3.0M ionic strength. After construction of a suitable apparatus, the experimental procedure and data reduction were computerized to perform a large number of titrations. Most measured pK{sub a}`s showed high reproducibility (the difference of pK{sub a} values observed between two experiments was less than 0.05). For IS = 0.1M, observed pK{sub a}`s agreed with literature values to within a few hundredths of a pH unit. Furthermore, the ionic-strength dependence of the pK{sub a}`s followed the trends reported in the literature, viz. pK{sub a} values decrease with increasing ionic strength until they reach a minimum at about IS = 0.5M. At still higher IS, pK{sub a}`s increase as the ionic strength rises to 3M. The known pK{sub a}`s of all titratable groups in a protein were used with the IST to give a first approximation of how the protein net charge varies with pH at high ionic strength. A comparison of the titration curves based on the IST with experimental lysozyme and {alpha}-chymotrypsin titration data indicates acceptable agreement at IS = 0.1M. However, comparison of measured and calculated titration curves at IS = 1M and IS = 3M indicates only quantitative agreement.

  17. Nucleation of lysozyme crystals under external electric and ultrasonic fields (United States)

    Nanev, Christo N.; Penkova, Anita


    Preferred orientation along c-axis of hen-egg-white lysozyme (HEWL) crystals has been observed in an external electric field. Besides, the HEWL crystals grew predominantly on the cathode side of the glass cell. These facts were explained on the basis of a concept for specific spatial distribution of the positive electric charges on the individual HEWL molecules, and thus attributed to the (preferred) orientation of individual HEWL molecules in the solution, under these conditions. Ultrasonic field redoubles the nucleation rate of HEWL crystals, but does not change the number of building units in the critical nucleus. Taking into account the intermolecular binding energy, we conclude that ultrasonic field accelerates nucleation due to breaking of the protein crystals.

  18. The effects of xylitol and sorbitol on lysozyme- and peroxidase-related enzymatic and candidacidal activities. (United States)

    Kim, Bum-Soo; Chang, Ji-Youn; Kim, Yoon-Young; Kho, Hong-Seop


    To investigate whether xylitol and sorbitol affect enzymatic and candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase system. Xylitol and sorbitol were added to hen egg-white lysozyme, bovine lactoperoxidase, glucose oxidase-mediated peroxidase, and whole saliva in solution and on hydroxyapatite surfaces. The enzymatic activities of lysozyme, peroxidase, and glucose oxidase-mediated peroxidase were determined by the turbidimetric method, the NbsSCN assay, and production of oxidized o-dianisidine, respectively. Candidacidal activities were determined by comparing colony forming units using Candida albicans ATCC strains 10231, 11006, and 18804. While xylitol and sorbitol did not affect the enzymatic activity of hen egg-white lysozyme both in solution and on hydroxyapatite surfaces, they did inhibit the enzymatic activity of salivary lysozyme significantly in solution, but not on the surfaces. Xylitol and sorbitol enhanced the enzymatic activities of both bovine lactoperoxidase and salivary peroxidase significantly in a dose-dependent manner in solution, but not on the surfaces. Sorbitol, but not xylitol, inhibited the enzymatic activity of glucose oxidase-mediated peroxidase significantly. Both xylitol and sorbitol did not affect candidacidal activities of hen egg-white lysozyme, the bovine lactoperoxidase system, or the glucose oxidase-mediated bovine lactoperoxidase system. Xylitol and sorbitol inhibited salivary lysozyme activity, but enhanced both bovine lactoperoxidase and salivary peroxidase activities significantly in solution. Xylitol and sorbitol did not augment lysozyme- and peroxidase-related candidacidal activities. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Solution Structure Determination of Proteins by Solution NMR: Application to a Envelope Protein, LAP2

    Institute of Scientific and Technical Information of China (English)


    @@ Recent advances in multidimensional NMR to obtain resonance assignments, interproton distance and torsion angle restraints, and restraints that characterize long range order, coupled with new methods of structure refinement, have permitted solution structures of proteins to be rapidly and quickly determined.

  20. Structure and stability of complex coacervate core micelles with lysozyme. (United States)

    Lindhoud, Saskia; Vries, Renko de; Norde, Willem; Stuart, Martien A Cohen


    Encapsulation of enzymes by polymers is a promising method to influence their activity and stability. Here, we explore the use of complex coacervate core micelles for encapsulation of enzymes. The core of the micelles consists of negatively charged blocks of the diblock copolymer PAA42PAAm417 and the positively charged homopolymer PDMAEMA150. For encapsulation, part of the positively charged homopolymer was replaced by the positively charged globular protein lysozyme. We have studied the formation, structure, and stability of the resulting micelles for three different mixing ratios of homopolymer and lysozyme: a system predominantly consisting of homopolymer, a system predominantly consisting of lysozyme, and a system where the molar ratio between the two positively charged molecules was almost one. We also studied complexes made of only lysozyme and PAA42PAAm417. Complex formation and the salt-induced disintegration of the complexes were studied using dynamic light-scattering titrations. Small-angle neutron scattering was used to investigate the structures of the cores. We found that micelles predominantly consisting of homopolymer are spherical but that complex coacervate core micelles predominantly consisting of lysozyme are nonspherical. The stability of the micelles containing a larger fraction of lysozyme is lower.


    Directory of Open Access Journals (Sweden)

    S. S.


    Full Text Available The aim of the research was the development of the method of lysozyme isolation from hen egg proteins. Lysozyme was isolated by differential heat denaturation of proteins with changing of the medium pH value, followed by neutralization, dialysis and additional purification by gel chromatography on Sephadex G-50. Activity was determined by bacteriolytic method (with Micrococcus lysodeikticus 4698 as a substrate. The enzyme purity and molecular mass were determined using SDS-electrophoresis and massspectrometry. The method of lysozyme isolation from hen egg proteins with the enzyme yield of 3.2 ± 0.2% and bacteriolytic activity of 22 025 ± 1 500 U/mg is modified. According to electrophoresis data, the isolated enzyme is characterized by high degree of purity (~95–98% and is comparable with lysozyme of AppliChem company by main physical and chemical characteristics. The obtaining product is stored in a crystalline form at low temperature (–24 оC for 9 months. The proposed method allows obtaining active and stable lysozyme with high purity from hen egg protein in laboratory conditions for the usage in biotechnology.

  2. Formation and seeding of amyloid fibrils from wild-type hen lysozyme and a peptide fragment from the beta-domain. (United States)

    Krebs, M R; Wilkins, D K; Chung, E W; Pitkeathly, M C; Chamberlain, A K; Zurdo, J; Robinson, C V; Dobson, C M


    Wild-type hen lysozyme has been converted from its soluble native state into highly organized amyloid fibrils. In order to achieve this conversion, conditions were chosen to promote partial unfolding of the native globular fold and included heating of low-pH solutions and addition of organic solvents. Two peptides derived from the beta-sheet region of hen lysozyme were also found to form fibrils very readily. The properties and morphologies of the amyloid fibrils formed by incubation either of the protein or the peptides are similar to those produced from the group of proteins associated with clinical amyloidoses. Fibril formation by hen lysozyme was substantially accelerated when aliquots of solutions in which fibrils of either one of the peptides or the full-length protein had previously formed were added to fresh solutions of the protein, revealing the importance of seeding in the kinetics of fibril formation. These findings support the proposition that the beta-domain is of particular significance in the formation of fibrils from the full-length protein and suggest similarities between the species giving rise to fibril formation and the intermediates formed during protein folding. Copyright 2000 Academic Press.

  3. Extraction of Thermodynamic Data from Ternary Diffusion Coefficients of Lysozyme Chloride in Water and Aqueous Na$_2$SO$_4$

    CERN Document Server

    Buzatu, D; Buzatu, F D; Albright, J G


    This paper presents, for ternary lysozyme-Na$_2$SO$_4$-water system, the thermodynamic data extracted from the measured values of four ternary diffusion coefficients and the Onsager reciprocal relations. The calculation for derivatives of solute chemical potentials with respect to solute molar concentrations was made using the method presented in \\cite{1}. This method is applicable to systems in which the molar concentration of one solute is very small compared to that of the other, like in our case. The approach is illustrated for the lysozyme chloride-Na$_2$SO$_4$-water system at 25$^o$ C, pH 4.5 and at 0.6 mM (8.6 mg/mL) lysozyme chloride and 0.1, 0.25, 0.5, 0.65, and 0.8 M Na$_2$SO$_4$ concentrations. The calculated solute chemical potential derivatives were used to compute the protein cation charge approximately. We also compute the diffusion Onsager coefficients $(L_{ij})_o$ for each composition at pH 4.5.

  4. Effects of purification on the crystallization of lysozyme (United States)

    Ewing, Felecia L.; Forsythe, Elizabeth L.; van der Woerd, Mark; Pusey, Marc L.


    We have additionally purified a commercial lysozyme preparation by cation exchange chromatography, followed by recrystallization. This material is 99.96% pure with respect to macromolecular impurities. At basic pH, the purified lysozyme gave only tetragonal crystals at 20°C. Protein used directly from the bottle, prepared by dialysis against distilled water, or which did not bind to the cation exchange column had considerably altered crystallization behavior. Lysozyme which did not bind to the cation exchange column was subsequently purified by size exclusion chromatography. This material gave predominately bundles of rod-shaped crystals with some small tetragonal crystals at lower pHs. The origin of the bundled rod habit was postulated to be a thermally dependent tetragonal ↔ orthorhombic change in the protein structure. This was subsequently ruled out on the basis of crystallization behavior and growth rate experiments. This suggests that heterogeneous forms of lysozyme may be responsible. These results demonstrate three classes of impurities: (1) small molecules, which may be removed by dialysis; (2) macromolecules, which are removable by chromatographic techniques; and (3) heterogeneous forms of the protein, which can be removed in this case by cation exchange chromatography. Of these, heterogeneous forms of the lysozyme apparently have the greatest affect on its crystallization behavior.

  5. Amyloid fibril formation and seeding by wild-type human lysozyme and its disease-related mutational variants. (United States)

    Morozova-Roche, L A; Zurdo, J; Spencer, A; Noppe, W; Receveur, V; Archer, D B; Joniau, M; Dobson, C M


    Wild-type human lysozyme and its two stable amyloidogenic variants have been found to form partially folded states at low pH. These states are characterized by extensive disruption of tertiary interactions and partial loss of secondary structure. Incubation of the proteins at pH 2.0 and 37 degrees C (Ile56Thr and Asp67His variants) or 57 degrees C (wild-type) results in the formation of large numbers of fibrils over several days of incubation. Smaller numbers of fibrils could be observed under other conditions, including neutral pH. These fibrils were analyzed by electron microscopy, Congo red birefringence, thioflavine-T binding, and X-ray fiber diffraction, which unequivocally show their amyloid character. These data demonstrate that amyloidogenicity is an intrinsic property of human lysozyme and does not require the presence of specific mutations in its primary structure. The amyloid fibril formation is greatly facilitated, however, by the introduction of "seeds" of preformed fibrils to the solutions of the variant proteins, suggesting that seeding effects could be important in the development of systemic amyloidosis. Fibril formation by wild-type human lysozyme is greatly accelerated by fibrils of the variant proteins and vice versa, showing that seeding is not specific to a given protein. The fact that wild-type lysozyme has not been found in ex vivo deposits from patients suffering from this disease is likely to be related to the much lower population of incompletely folded states for the wild-type protein compared to its amyloidogenic variants under physiological conditions. These results support the concept that the ability to form amyloid is a generic property of proteins, but one that is mitigated against in a normally functioning organism. Copyright 2000 Academic Press.

  6. A highly amyloidogenic region of hen lysozyme. (United States)

    Frare, Erica; Polverino De Laureto, Patrizia; Zurdo, Jesús; Dobson, Christopher M; Fontana, Angelo


    Amyloid fibrils obtained after incubating hen egg-white lysozyme (HEWL) at pH 2.0 and 65 degrees C for extended periods of time have been found to consist predominantly of fragments of the protein corresponding to residues 49-100, 49-101, 53-100 and 53-101, derived largely from the partial acid hydrolysis of Asp-X peptide bonds. These internal fragments of HEWL encompass part of the beta-domain and all the residues forming the C-helix in the native protein, and contain two internal disulfide bridges Cys64-Cys80 and Cys76-Cys94. The complementary protein fragments, including helices A, B and D of the native protein, are not significantly incorporated into the network of fibrils, but remain largely soluble, in agreement with their predicted lower propensities to aggregate. Further analysis of the properties of different regions of HEWL to form amyloid fibrils was carried out by studying fragments produced by limited proteolysis of the protein by pepsin. Here, we show that only fragment 57-107, but not fragment 1-38/108-129, is able to generate well-defined amyloid fibrils under the conditions used. This finding is of particular importance, as the beta-domain and C-helix of the highly homologous human lysozyme have been shown to unfold locally in the amyloidogenic variant D67H, which is associated with the familial cases of systemic amyloidosis linked to lysozyme deposition. The identification of the highly amyloidogenic character of this region of the polypeptide chain provides strong support for the involvement of partially unfolded species in the initiation of the aggregation events that lead to amyloid deposition in clinical disease.

  7. Crystallization of Hevamine, an Enzyme with Lysozyme/Chitinase Activity from Hevea brasiliensis Latex

    NARCIS (Netherlands)



    Hevamine, an enzyme with both lysozyme and chitinase activity, was isolated and purified from Hevea brasiliensis (rubber tree) latex. The enzyme (molecular weight 29,000) is homologous to certain “pathogenesis-related” proteins from plants, but not to hen egg-white or phage T4 lysozyme. To

  8. Production of active lysozyme films by matrix assisted pulsed laser evaporation at 355 nm

    DEFF Research Database (Denmark)

    Purice, Andreea; Schou, Jørgen; Kingshott, P.;


    Thin lysozyme films have been produced in a dry environment by MAPLE (matrix assisted pulsed laser evaporation) from a water ice matrix irradiated by laser light at 355 nm above the absorption threshold of the protein. A significant part of the lysozyme molecules are transferred to the film without...

  9. Crystallization of Hevamine, an Enzyme with Lysozyme/Chitinase Activity from Hevea brasiliensis Latex

    NARCIS (Netherlands)



    Hevamine, an enzyme with both lysozyme and chitinase activity, was isolated and purified from Hevea brasiliensis (rubber tree) latex. The enzyme (molecular weight 29,000) is homologous to certain “pathogenesis-related” proteins from plants, but not to hen egg-white or phage T4 lysozyme. To investiga

  10. The Plasmid-Encoded Regulator Activates Factors Conferring Lysozyme Resistance on Enteropathogenic Escherichia coli Strains▿ (United States)

    Salinger, Nina; Kokona, Bashkim; Fairman, Robert; Okeke, Iruka N.


    We demonstrate that enhanced lysozyme resistance of enteropathogenic Escherichia coli requires the plasmid-encoded regulator, Per, and is mediated by factors outside the locus for enterocyte effacement. EspC, a Per-activated serine protease autotransporter protein, conferred enhanced resistance on nonpathogenic E. coli, and a second Per-regulated, espC-independent lysozyme resistance mechanism was identified. PMID:18997020

  11. The plasmid-encoded regulator activates factors conferring lysozyme resistance on enteropathogenic Escherichia coli strains. (United States)

    Salinger, Nina; Kokona, Bashkim; Fairman, Robert; Okeke, Iruka N


    We demonstrate that enhanced lysozyme resistance of enteropathogenic Escherichia coli requires the plasmid-encoded regulator, Per, and is mediated by factors outside the locus for enterocyte effacement. EspC, a Per-activated serine protease autotransporter protein, conferred enhanced resistance on nonpathogenic E. coli, and a second Per-regulated, espC-independent lysozyme resistance mechanism was identified.

  12. Thermodynamic Exploration of Eosin-Lysozyme Binding: A Physical Chemistry and Biochemistry Laboratory Experiment (United States)

    Huisman, Andrew J.; Hartsell, Lydia R.; Krueger, Brent P.; Pikaart, Michael J.


    We developed a modular pair of experiments for use in the undergraduate physical chemistry and biochemistry laboratories. Both experiments examine the thermodynamics of the binding of a small molecule, eosin Y, to the protein lysozyme. The assay for binding is the quenching of lysozyme fluorescence by eosin through resonant energy transfer. In…

  13. Surface morphology of thin lysozyme films produced by matrix-assisted pulsed laser evaporation (MAPLE)

    DEFF Research Database (Denmark)

    Purice, Andreea; Schou, Jørgen; Pryds, Nini;


    Thin films of the protein, lysozyme, have been deposited by the matrix-assisted pulsed laser evaporation (MAPLE) technique. Frozen targets of 0.3-1.0 wt.% lysozyme dissolved in ultrapure water were irradiated by laser light at 355 mn with a fluence of 2 J/cm(2). The surface quality of the thin ly...

  14. Non-covalent conjugation of CdTe QDs with lysozyme binding DNA for fluorescent sensing of lysozyme in complex biological sample. (United States)

    Li, Shujia; Gao, Zhidan; Shao, Na


    Water-soluble cysteamine (CA) capped CdTe quantum dots (QDs) conjugated with lysozyme binding DNA (LBD) was constructed for luminescent sensing of lysozyme by forming a ternary self-assembly complex. Addition of negatively charged lysozyme binding DNA to the positively charged CA capped CdTe QDs buffer solution (Tris-HCl pH 7.4) could lead to the formation of QDs-LBD complex through electrostatic interactions. Once lysozyme was introduced into the CdTe QDs-LBD system, it could bind specifically with the QDs-LBD complex, resulting in fluorescence emission enhancement of the QDs due to the surface inert of QDs. At a given amount of LBD and CdTe QDs (LBD: QDs=2: 1), the fluorescence intensity enhancement of QDs was linear with lysozyme concentration over the range of 8.9-71.2 nM, with a detection limit of 4.3 nM. Due to the specific binding of LBD with lysozyme, this approach displayed high selectivity for lysozyme recognition. The sensing mechanism was confirmed by DLS and zeta potential measurement, and agarose gel electrophoresis experiment. Furthermore, the proposed CA-capped CdTe QDs-LBD sensor was applied to lysozyme detection in mouse serum and human morning urine samples, which showed high sensitivity and selectivity in the complex biological sample.

  15. Study of Growth Mechanism of Lysozyme Crystal by Batch Crystallization Method

    Institute of Scientific and Technical Information of China (English)

    Hai Liang CUI; Yong YU; Wan Chun CHEN; Qi KANG


    The lysozyme crystals were made by batch crystallization method and the distribution of aggregate in solution were measured by dynamic light scattering. The results showed that the dimension of aggregate increased with the increase of the concentration of lysozyme and NaC1,lysozyme molecules aggregated gradually in solution and finally arrived at balance each other.The higher the concentrations of lysozyme and NaC1 were, the faster the growth rate of (110) face was. The growth rates of lysozyme crystal were obtained by a Zeiss microscope, and the effective surface energy (α) of growing steps were calculated about 4.01×l0-8 according to the model of multiple two-dimensional nucleation mechanism.


    Directory of Open Access Journals (Sweden)

    Dekina S. S.


    Full Text Available The study of non-covalent immobilized lysozyme, as well as physico-chemical and biochemical properties of obtained mucoadhesive gel was the aim of the research. Lysozyme activity was determined by bacteriolytic method (Micrococcus lysodeikticus cells acetone powder was a substrate. Lysozyme immobilization was conducted by the method of entrapment in gel. Enzyme carrier interaction was studied by viscometric, spectrophotometric and spectrofluorimetric methods. Mucoadhesive gel with immobilized lysozyme, possessing antiinflammatory and antimicrobial activities, was prepared. Due to immobilization, protein-polymer complex with the original enzymatic activity was formed. The product is characterized by high mucoadhesive properties, quantitative retaining of protein and bacteriolytic activity, prolonged release of the enzyme, improved biochemical characteristics (extended pH-activity profile, stability in acidic medium and during storage for 2 years, and it is perspective for further studies. The proposed method for lysozyme immobilization in the carboxymethyl cellulose sodium salt gel allows to obtain a stable, highly efficient product, with high adhesive properties for attachment to the mucous membranes, that is promising for use in biomedicine.

  17. AFM Studies of Salt Concentration Effects on the (110) Surface Structure of Tetragonal Lysozyme Crystals (United States)

    Pusey, Marc Lee; Gorti, Sridhar; Forsythe, Elizabeth; Konnert, John


    Previous high resolution AFM studies of the (110) surface of tetragonal chicken egg white lysozyme crystals had shown that only one of two possible molecular surfaces is present, those constituting the completed 43 helices. These suggested that the crystal growth process was by the solution-phase assembly of the growth units, which then attach to the surface. However, the best fit for the imaged surfaces, vs. those predicted based upon the bulk crystallographic coordinates, were obtained when the packing about the 43 helices was "tightened up", while maintaining the underlying crystallographic unit cell spacing. This results in a widening of the gap between adjacent helices, and the top- most layer(s) may no longer be in contact. We postulated that the tightened packing about the helices is a result of the high salt concentrations in the bulk solution, used to crystallize the protein, driving hydrophobic interactions. Once the crystal surface is sufficiently buried by subsequent growth layers the ratio of salt to protein molecules decreases and the helices relax to their bulk crystallographic coordinates. The crystal surface helix structure is thus a reflection of the solution structure, and the tightness of the packing about the 43 helices would be a function of the bulk salt concentration. AFM images of the (110) surface of tetragonal lysozyme crystals grown under low (2%) and high (5%) NaCl concentrations reveal differences in the packing about the 43 helices consistent with the above proposal.

  18. Effect of cholesterol on the properties of spray-dried lysozyme-loaded liposomal powders. (United States)

    Charnvanich, Dusadee; Vardhanabhuti, Nontima; Kulvanich, Poj


    The influence of cholesterol (Chol) in the liposomal bilayer on the properties of inhalable protein-loaded liposomal powders prepared by spray-drying technique was investigated. Lysozyme (LSZ) was used as a model protein. Feed solution for spray drying was prepared by direct mixing of aqueous solution of LSZ with mannitol solution and empty liposome dispersions composed of hydrogenated phosphatidylcholine and Chol at various molar ratios. The spray-dried powders were characterized with respect to morphology, thermal property, and crystallinity using scanning electron microscopy, differential scanning calorimetry, and X-ray diffraction, respectively. Most formulations gave slightly aggregated, spherical particles, and percentage yields of the spray-dried powders decreased with increasing Chol content. Degree of particle aggregation depended on the powder composition. The powders spontaneously formed liposomes which efficiently entrapped LSZ after reconstitution with HEPES buffered saline (HBS) at 37 degrees C. Lysozyme entrapment efficiency and size distribution of the reconstituted liposomes were evaluated after the powders were reconstituted with HBS. Increasing Chol content resulted in a decrease in size of the reconstituted liposomes and an increase in entrapment efficiency of LSZ. These results correlated with thermal behaviors of the reconstituted liposomes. Biological activity of LSZ was not affected by the spray-drying process. It was also demonstrated that LSZ-loaded liposomal powders could be produced without the need to preload the LSZ into liposomes prior to spray-drying process.

  19. Lysozyme in water-acetonitrile mixtures: Preferential solvation at the inner edge of excess hydration (United States)

    Sirotkin, Vladimir A.; Kuchierskaya, Alexandra A.


    Preferential solvation/hydration is an effective way for regulating the mechanism of the protein destabilization/stabilization. Organic solvent/water sorption and residual enzyme activity measurements were performed to monitor the preferential solvation/hydration of hen egg-white lysozyme at high and low water content in acetonitrile at 25 °C. The obtained results show that the protein destabilization/stabilization depends essentially on the initial hydration level of lysozyme and the water content in acetonitrile. There are three composition regimes for the dried lysozyme. At high water content, the lysozyme has a higher affinity for water than for acetonitrile. The residual enzyme activity values are close to 100%. At the intermediate water content, the dehydrated lysozyme has a higher affinity for acetonitrile than for water. A minimum on the residual enzyme activity curve was observed in this concentration range. At the lowest water content, the organic solvent molecules are preferentially excluded from the dried lysozyme, resulting in the preferential hydration. The residual catalytic activity is ˜80%, compared with that observed after incubation in pure water. Two distinct schemes are operative for the hydrated lysozyme. At high and intermediate water content, lysozyme is preferentially hydrated. However, in contrast to the dried protein, at the intermediate water content, the initially hydrated lysozyme has the increased preferential hydration parameters. At low water content, the preferential binding of the acetonitrile molecules to the initially hydrated lysozyme was detected. No residual enzyme activity was observed in the water-poor acetonitrile. Our data clearly show that the initial hydration level of the protein macromolecules is one of the key factors that govern the stability of the protein-water-organic solvent systems.

  20. Complex coacervate core micelles with a lysozyme-modified corona. (United States)

    Danial, Maarten; Klok, Harm-Anton; Norde, Willem; Stuart, Martien A Cohen


    This paper describes the preparation, characterization, and enzymatic activity of complex coacervate core micelles (C3Ms) composed of poly(acrylic acid) (PAA) and poly(N-methyl-2-vinyl pyridinium iodide)-b-poly(ethylene oxide) (PQ2VP-PEO) to which the antibacterial enzyme lysozyme is end-attached. C3Ms were prepared by polyelectrolyte complex formation between PAA and mixtures containing different ratios of aldehyde and hydroxyl end-functionalized PQ2VP-PEO. This resulted in the formation of C3Ms containing 0-40% (w/w) of the aldehyde end-functionalized PQ2VP-PEO block copolymer (PQ2VP-PEO-CHO). Chemical conjugation of lysozyme was achieved via reductive amination of the aldehyde groups, which are exposed at the surface of the C3M, with the amine groups present in the side chains of the lysine residues of the protein. Dynamic and static light scattering indicated that the conjugation of lysozyme to C3Ms prepared using 10 and 20% (w/w) PQ2VP-PEO-CHO resulted in the formation of unimicellar particles. Multimicellar aggregates, in contrast, were obtained when lysozyme was conjugated to C3Ms prepared using 30 or 40% (w/w) PQ2VP-PEO-CHO. The enzymatic activity of the unimicellar lysozyme-C3M conjugates toward the hydrolysis of the bacterial substrate Micrococcus lysodeikticus was comparable to that of free lysozyme. For the multimicellar particles, in contrast, significantly reduced enzymatic rates of hydrolysis, altered circular dichroism, and red-shifted tryptophan fluorescence spectra were measured. These results are attributed to the occlusion of lysozyme in the interior of the multimicellar conjugates.

  1. [Lysozyme in the treatment of juvenile laryngeal papillomatosis. A new concept in its etiopathogenesis]. (United States)

    Altamar-Ríos, J


    The A. inform about the results achieved with lysozyme chlorhydrate in the treatment of 15 patients with juvenile laryngeal papillomatosis. The lysozyme is an electropositive enzyme which synthesis is related to the degree of proteins and vitamin B complex ingestion. Lysozyme is a component of the immunitary inespecific system, serving to prevent against HPV-DNA at the level of the secretory film of the mucociliary apparatus of the respiratory mucous membrane. Furthermore, lysozyme hydrolyzes the mucopolysaccharide of the connective tissue and inhibits the virus-DNA replication. 100-300 mgr daily during 30-60 days simultaneously with hyperproteic diet and vitamin B complex (after correction of the nutrimental deficiencies) brought about the evanishment of papillomatosis. The A. suggest that the predisposition to infection by virus DNA is primarily of immunitary origin, because of lysozyme deficiency, and secondary due to a low intake of proteins and vitamin B complex.

  2. Fabrication and characterization of mesoscale protein patterns using atomic force microscopy (AFM) (United States)

    Gao, Pei


    A versatile AFM local oxidation lithography was developed for fabricating clean protein patterns ranging from nanometer to sub-millimeter scale on octadecyltrichlorosilane (OTS) layer of Si (100) wafer. This protein patterning method can generate bio-active protein pattern with a clean background without the need of the anti-fouling the surface or repetitive rinsing. As a model system, lysozyme protein patterns were investigated through their binding reactions with antibodies and aptamers by AFM. Polyclonal anti-lysozyme antibodies and anti-lysozyme aptamer are found to preferentially bind to the lysozyme molecules on the edge of a protein pattern before their binding to the interior ones. It was also demonstrated that the topography of the immobilized protein pattern affects the antibody binding direction. We found that the anti-lysozyme antibodies binding to the edge lysozyme molecules on the half-buried pattern started from the top but the binding on the extruded pattern started from the side because of their different spatial accessibility. In addition, after incubating lysozyme pattern with anti-lysozyme aptamer in buffer solution for enough long time, some fractal-shaped aptamer fibers with 1-6nm high and up to tens of micrometers long were formed by the self-assembling of aptamer molecules on the surface. The aptamer fibers anchor specifically on the edge of protein patterns, which originates from the biospecific recognition between the aptamer and its target protein. Once these edge-bound fibers have formed, they can serve as scaffolds for further assembly processes. We used these aptamer fibers as templates to fabricate palladium and streptavidin nanowires, which anchored on the pattern edges and never cross over or collapse over each other. The aptamer fiber scaffold potentially can lead to an effective means to fabricate and interface nanowires to existing surface patterns. KEYWORDS: Atomic Force Microscopy (AFM), Protein Patterns, Lysozyme, Aptamer

  3. New views on foams from protein solutions

    NARCIS (Netherlands)

    Wierenga, P.A.; Gruppen, H.


    The stabilization of foam by proteins has been mostly studied in relation to the food industry. The main aim of the research is to understand the relation between proteins used and the product properties. The molecular properties of proteins and their foam forming and stabilizing properties are typi

  4. Directed destabilization of lysozyme in protic ionic liquids reveals a compact, low energy, soluble, reversibly-unfolding (pre-fibril) state

    CERN Document Server

    Byrne, Nolene; Angell, C Austen


    Recent demonstrations of extraordinary stabilization of proteins in mobile protic [1] and aprotic [2] ionic liquid solutions at ambient temperatures have raised hopes of new biopreservation and drug transportation technologies. Here we examine the relation of folded protein stability to the state of the transferred proton [1], as determined by the N-H proton chemical shift, d(N-H). We identify a range of d(N-H) in which the unfolded lysozyme refolds 97%. Exceeding the stability range in the acid direction leads to the sudden formation and stabilization of a small, soluble, amyloid form of lysozyme which has its own stability range and which can again unfold/refold many times before an irreversible process, fibrillization, occurs. The tightly bound amyloid form of the lysozyme molecule, identified by circular dichroism spectra and dynamic light scattering, must be of very low energy since the unfolding process absorbs almost three times the enthalpy of normal lysozyme unfolding. alpha-lactalbumin shows similar...

  5. Inclusion of mPRISM potential for polymer-induced protein interactions enables modeling of second osmotic virial coefficients in aqueous polymer-salt solutions. (United States)

    Herhut, Marcel; Brandenbusch, Christoph; Sadowski, Gabriele


    The downstream processing of therapeutic proteins is a challenging task. Key information needed to estimate applicable workup strategies (e.g. crystallization) are the interactions of the proteins with other components in solution. This information can be deduced from the second osmotic virial coefficient B22 , measurable by static light scattering. Thermodynamic models are very valuable for predicting B22 data for different process conditions and thus decrease the experimental effort. Available B22 models consider aqueous salt solutions but fail for the prediction of B22 if an additional polymer is present in solution. This is due to the fact that depending on the polymer concentration protein-protein interactions are not rectified as assumed within these models. In this work, we developed an extension of the xDLVO model to predict B22 data of proteins in aqueous polymer-salt solutions. To show the broad applicability of the model, lysozyme, γ-globulin and D-xylose ketol isomerase in aqueous salt solution containing polyethylene glycol were considered. For all proteins considered, the modified xDLVO model was able to predict the experimentally observed non-monotonical course in B22 data with high accuracy. When used in an early stage in process development, the model will contribute to an efficient and cost effective downstream processing development.

  6. Towards Inferring Protein Interactions: Challenges and Solutions

    Directory of Open Access Journals (Sweden)

    Ji Xiang


    Full Text Available Discovering interacting proteins has been an essential part of functional genomics. However, existing experimental techniques only uncover a small portion of any interactome. Furthermore, these data often have a very high false rate. By conceptualizing the interactions at domain level, we provide a more abstract representation of interactome, which also facilitates the discovery of unobserved protein-protein interactions. Although several domain-based approaches have been proposed to predict protein-protein interactions, they usually assume that domain interactions are independent on each other for the convenience of computational modeling. A new framework to predict protein interactions is proposed in this paper, where no assumption is made about domain interactions. Protein interactions may be the result of multiple domain interactions which are dependent on each other. A conjunctive norm form representation is used to capture the relationships between protein interactions and domain interactions. The problem of interaction inference is then modeled as a constraint satisfiability problem and solved via linear programing. Experimental results on a combined yeast data set have demonstrated the robustness and the accuracy of the proposed algorithm. Moreover, we also map some predicted interacting domains to three-dimensional structures of protein complexes to show the validity of our predictions.

  7. Mapping the solid-state properties of crystalline lysozyme during pharmaceutical unit-operations. (United States)

    Mohammad, Mohammad Amin; Grimsey, Ian M; Forbes, Robert T


    Bulk crystallisation of protein therapeutic molecules towards their controlled drug delivery is of interest to the biopharmaceutical industry. The complexity of biotherapeutic molecules is likely to lead to complex material properties of crystals in the solid state and to complex transitions. This complexity is explored using batch crystallised lysozyme as a model. The effects of drying and milling on the solid-state transformations of lysozyme crystals were monitored using differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), FT-Raman, and enzymatic assay. XRPD was used to characterise crystallinity and these data supported those of crystalline lysozyme which gave a distinctive DSC thermogram. The apparent denaturation temperature (Tm) of the amorphous lysozyme was ∼201 °C, while the Tm of the crystalline form was ∼187 °C. Raman spectra supported a more α-helix rich structure of crystalline lysozyme. This structure is consistent with reduced cooperative unit sizes compared to the amorphous lysozyme and is consistent with a reduction in the Tm of the crystalline form. Evidence was obtained that milling also induced denaturation in the solid-state, with the denatured lysozyme showing no thermal transition. The denaturation of the crystalline lysozyme occurred mainly through its amorphous form. Interestingly, the mechanical denaturation of lysozyme did not affect its biological activity on dissolution. Lysozyme crystals on drying did not become amorphous, while milling-time played a crucial role in the crystalline-amorphous-denatured transformations of lysozyme crystals. DSC is shown to be a key tool to monitor quantitatively these transformations.

  8. Lysozyme Resistance in Streptococcus suis Is Highly Variable and Multifactorial

    NARCIS (Netherlands)

    Wichgers, P.J.; Weeghel, van C.; Rebel, J.M.J.; Smits, M.A.; Putten, van J.P.M.; Smith, H.E.


    Background Streptococcus suis is an important infectious agent for pigs and occasionally for humans. The host innate immune system plays a key role in preventing and eliminating S. suis infections. One important constituent of the innate immune system is the protein lysozyme, which is present in a v

  9. Slow internal protein dynamics in solution (United States)

    Biehl, R.; Richter, D.


    Large-scale domain dynamics in proteins are found when flexible linkers or hinges connect domains. The related conformational changes are often related to the function of the protein, for example by arranging the active center after substrate binding or allowing transport and release of products. The adaptation of a specific active structure is referred to as ‘induced fit’ and is challenged by models such as ‘conformational sampling’. Newer models about protein function include some flexibility within the protein structure or even internal dynamics of the protein. As larger domains contribute to the configurational changes, the timescale of the involved motions is slowed down. The role of slow domain dynamics is being increasingly recognized as essential to understanding the function of proteins. Neutron spin echo spectroscopy (NSE) is a technique that is able to access the related timescales from 0.1 up to several hundred nanoseconds and simultaneously covers the length scale relevant for protein domain movements of several nanometers distance between domains. Here we focus on these large-scale domain fluctuations and show how the structure and dynamics of proteins can be assessed by small-angle neutron scattering and NSE.

  10. An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates. (United States)

    Padlan, Camille S; Malashkevich, Vladimir N; Almo, Steve C; Levy, Matthew; Brenowitz, Michael; Girvin, Mark E


    RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid-protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme. The resultant 59-nucleotide compact aptamer (Lys1.2minE) retains nanomolar binding affinity and the ability to inhibit lysozyme's catalytic activity. Our 2.0-Å crystal structure of the aptamer-protein complex reveals a helical stem stabilizing two loops to form a protein binding platform that binds lysozyme distal to the catalytic cleft. This structure along with complementary solution analyses illuminate a novel protein-nucleic acid interface; (1) only 410 Å(2) of solvent accessible surface are buried by aptamer binding; (2) an unusually small fraction (∼18%) of the RNA-protein interaction is electrostatic, consistent with the limited protein phosphate backbone contacts observed in the structure; (3) a single Na(+) stabilizes the loops that constitute the protein-binding platform, and consistent with this observation, Lys1.2minE-lysozyme complex formation takes up rather than displaces cations at low ionic strength; (4) Lys1.2minE inhibits catalysis of large cell wall substrates but not catalysis of small model substrates; and (5) the helical stem of Lys1.2minE can be shortened to four base pairs (Lys1.2minF) without compromising binding affinity, yielding a 45-nucleotide aptamer whose structure may be an adaptable protein binding platform.

  11. Computational Solutions to the Protein Folding Problem, (United States)


    the chemical construction of proteins. The use of knowledge such as the Ramachandran plot ( Lehninger 1970) which specifies the possible values for...Note that similar information has been developed for proteins using the Ramachandran plot ( Lehninger 1970). B. The Conformation of Heptane Heptane is an...Boston. 60 Lehninger , A.L. (1970), Biochemistqy: The Molecular Basis of Cell Structure and Func- tion, Worth Publishers, New York. Maranas, C.D. and

  12. Protein conformation in solution by three-dimensional fluorescence spectrometry

    Institute of Scientific and Technical Information of China (English)

    鄢远; 许金钩; 陈国珍


    The conformations of bovine serum albumin (USA) and egg albumin (EA) in solution and their conformation changes under different conditions were studied by using three-dimensional fluorescence spectrometry (TDFS) such as three-dimensional fluorescence (TDF) spectra and three-dimensional fluorescence polarization (TDFP) spectra with tryptophan residues in protein molecules as an intrinsic fluorescent probe. The results show that the microenvironment of tryptophan residues of protein molecules in various solutions can be directly indicated and TDFS is an effective tool for studying protein conformation in solution. Meantime, some valuable results were obtained.

  13. Aggregation behavior of novel heptamethine cyanine dyes upon their binding to native and fibrillar lysozyme. (United States)

    Vus, Kateryna; Tarabara, Ulyana; Kurutos, Atanas; Ryzhova, Olga; Gorbenko, Galyna; Trusova, Valeriya; Gadjev, Nikolai; Deligeorgiev, Todor


    Two newly synthesized symmetrical heptamethine cyanine dyes, AK7-5 and AK7-6, absorbing in the region of low autofluorescence of biological samples, have been tested for their ability to detect proteins aggregated into amyloid fibrils. In aqueous solution these probes possess three absorption bands corresponding to the monomer, dimer and H-aggregate species. The association of the dye with fibrillar lysozyme was followed by the enhancement of the monomer band and the reduction of the H-band. The absorption spectra measured at various fibril concentrations were analyzed in terms of the model allowing for the shift of equilibria between various dye species due to the binding of monomers and dimers of AK7-5 and AK7-6 to amyloid fibrils. The association constants and stoichiometries of the dye-fibril complexation have been evaluated. In contrast to fibrillar lysozyme, the native protein brought about strong J-aggregate formation accompanied by a marked drop in the absorbance of the dye monomer species. Quantum chemical calculations and simple docking studies showed that AK7-5 and AK7-6 monomers can bind to the grooves, running parallel to the fibril axis. Due to their ability to distinguish between the native and fibrillar protein states, the novel cyanines are recommended as complementary to existing amyloid markers.

  14. 家蝇天蚕素-人溶菌酶融合蛋白的生物信息学分析%Bioinformatic analysis of Musca domestica cecropin-human lysozyme fusion protein

    Institute of Scientific and Technical Information of China (English)

    卢雪梅; 金小宝; 朱家勇; 黄演婷


    目的 利用生物信息学方法分析家蝇天蚕素-人溶菌酶融合基因推导的氨基酸序列.方法 用ProtParam Tool、CDD、ProtScal、sopma等软件对其理化性质、疏水性/亲水性、信号肽、功能结构域及蛋白质二级结构等重要参数进行预测.结果 家蝇天蚕素-人溶菌酶由l87个氨基酸组成,预测相对分子质量为20 131.7,理论等电点(pI)为9.69,分子式为C862 H1375 N277 O260 S11;半衰期预测结果显示其利于基因工程表达.融合蛋白的氨基酸序列含有天蚕素家族和溶菌酶家族二者的保守结构域,二级结构主要由α-螺旋、β-折叠、β-转角和无规则卷曲组成.结论 分析结果为家蝇天蚕素-人溶菌酶融合基因的原核表达及表达产物的生物学功能研究奠定了基础.%Objective To analyze the amino acid sequences of Musca domestica cecropin-human lysozyme (Mdc-HLY) fusion protein by bioinformatics analysis. Methods The physical-chemical properties, hydrophobicity or hydrophilicity, the signal peptide, the conserved domains and protein secondary structure of Mdc-HLY were analyzed by ProtParam Tool, CDD, ProtScal, sopma et al. Results Mdc-HLY was cationic molecules and was composed of 187 amino acids,with molecular weight of 20 131.7,theoretical PI of 9.69,the structural formula of C862H1375N277O260S11. The fusion protein included the conserved domains of both Musca domestica cecropin and human lysozyme. Instability index classified the protein as stable, and conducive to its expression using genetic engineering. The secondary structure of Mdc-HLY contained α-helix,β-sheet ,β-tum and random coil. Conclusion These results may provide foundation for further study on the expression and biological activity of Mdc-HLY.

  15. Interaction between lysozyme and procyanidin: multilevel structural nature and effect of carbohydrates. (United States)

    Liang, Miao; Liu, Rui; Qi, Wei; Su, Rongxin; Yu, Yanjun; Wang, Libing; He, Zhimin


    The interaction of procyanidins with proteins has aroused extensive attention due to its important relationship with the bioavailability and astringent property of polyphenols. In the present work, we have investigated the interactions of lysozyme with procyanidin dimer (B3) using various biophysical approaches, which aims to provide insights into the mechanism of protein/polyphenol aggregation. Procyanidin B3 spontaneously binds lysozyme, inducing the multilevel structural changes in lysozyme and the formation of insoluble complexes. The relationship between lysozyme aggregation and the loss of enzymatic activity was monitored using dynamic light scattering and fluorescence quenching. The influences of two carbohydrates (gum arabic and sucrose) on lysozyme/B3 aggregation were also studied. Gum arabic effectively inhibited the formation of insoluble aggregates, but was unable to restore the fluorescence and activity of lysozyme. However, sucrose concomitantly decreased the aggregate size with the recovery of fluorescence and lysozyme activity. These results proposed two probable mechanisms by which these two carbohydrates inhibit protein/polyphenol aggregation.

  16. Covalent immobilization of lysozyme on ethylene vinyl alcohol films for nonmigrating antimicrobial packaging applications. (United States)

    Muriel-Galet, V; Talbert, J N; Hernandez-Munoz, P; Gavara, R; Goddard, J M


    The objective of this study was to develop a new antimicrobial film, in which lysozyme was covalently attached onto two different ethylene vinyl alcohol copolymers (EVOH 29 and EVOH 44). The EVOH surface was modified with UV irradiation treatment to generate carboxylic acid groups, and lysozyme was covalently attached to the functionalized polymer surface. Surface characterization of control and modified films was performed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and dye assay. The value of protein loading after attachment on the surface was 8.49 μg protein/cm(2) and 5.74 μg protein/cm(2) for EVOH 29 and EVOH 44, respectively, after 10 min UV irradiation and bioconjugation. The efficacy of the EVOH-lysozyme films was assessed using Micrococcus lysodeikticus. The antimicrobial activity of the films was tested against Listeria monocytogenes and was similar to an equivalent amount of free enzyme. The reduction was 1.08 log for EVOH 29-lysozyme, 0.95 log for EVOH 44-lysozyme, and 1.34 log for free lysozyme. This work confirmed the successful use of lysozyme immobilization on the EVOH surface for antimicrobial packaging.

  17. Protein folding, protein structure and the origin of life: Theoretical methods and solutions of dynamical problems (United States)

    Weaver, D. L.


    Theoretical methods and solutions of the dynamics of protein folding, protein aggregation, protein structure, and the origin of life are discussed. The elements of a dynamic model representing the initial stages of protein folding are presented. The calculation and experimental determination of the model parameters are discussed. The use of computer simulation for modeling protein folding is considered.

  18. Non-monotonic course of protein solubility in aqueous polymer-salt solutions can be modeled using the sol-mxDLVO model. (United States)

    Herhut, Marcel; Brandenbusch, Christoph; Sadowski, Gabriele


    Protein purification is often performed using cost-intensive chromatographic steps. To discover economic alternatives (e.g., crystallization), knowledge on protein solubility as a function of temperature, pH, and additives in solution as well as their concentration is required. State-of-the-art models for predicting protein solubility almost exclusively consider aqueous salt systems, whereas "salting-in" and "salting-out" effects induced by the presence of an additional polymer are not considered. Thus, we developed the sol-mxDLVO model. Using this newly developed model, protein solubility in the presence of one salt and one polymer, especially the non-monotonic course of protein solubility, could be predicted. Systems considered included salts (NaCl, Na-p-Ts, (NH(4))(2) SO(4)) and the polymer polyethylene glycol (MW: 2000 g/mol, 12000 g/mol) and proteins lysozyme from chicken egg white (pH 4 to 5.5) and D-xylose ketol-isomerase (pH 7) at 298.15 K. The results show that by using the sol-mxDLVO model, protein solubility in polymer-salt solutions can be modeled in good agreement with the experimental data for both proteins considered. The sol-mxDLVO model can describe the non-monotonic course of protein solubility as a function of polymer concentration and salt concentration, previously not covered by state-of-the-art models.

  19. Effects of Chitosan, Lysozyme and Oregano Oil on Antimicrobial Activity of Soy Protein Isolate Film%壳聚糖、溶菌酶和牛至油对大豆分离蛋白膜抑菌效果的影响

    Institute of Scientific and Technical Information of China (English)

    杜会云; 赵寿经; 王新伟; 马中苏; 吴晓艳; 张艳平


    采用双倍稀释法研究壳聚糖、溶菌酶和牛至油对大肠杆菌和金黄色葡萄球菌的最小抑菌浓度.采用抑菌圈法研究添加天然抑菌剂壳聚糖、溶菌酶和牛至油的大豆分离蛋白膜对大肠杆菌、金黄色葡萄球菌、酿酒酵母和黑曲霉的抑菌效果.结果表明,添加壳聚糖、溶菌酶和牛至油的大豆分离蛋白膜对大肠杆菌、金黄色葡萄球菌、酿酒酵母和黑曲霉均有抑制作用.对大肠杆菌和金黄色葡萄球菌抑制效果为:牛至油>壳聚糖>溶菌酶.对酿酒酵母的抑制效果为:壳聚糖>牛至油>溶菌酶;对黑曲零的抑制效果为:牛至油>壳聚糖>溶菌酶.因此,添加壳聚糖、溶菌酶和牛至油的大豆分离蛋白膜具有较好的抑菌效果和应用前景.%Double dilution method was used to determine minimum inhibitory concentration of chitosan, lysozyme and oregano oil against Escherichia coli and Staphyloccocus aureus.Inhibition zone method was used to determine antimicrobial effect of soy protein isolate films enriched with chitosan, lysozyme and oregano oil against E.coli, S.aureus,Saccharomyces cerevisiae and Aspergillus niger.Results indicated that soy protein isolate films enriched with chitosan,lysozyme and oregano oil could inhibit the growth of E.coli, S.aureus, S.cerevisiae and A.niger.The inhibitory effect on E.coli and S.aureus: oregano oil > chitosan > lysozyme.The inhibitory effect on S.cerevisiae: chitosan > oregano oil > lysozyme; the inhibitory effect on A.niger: oregano oil > chitosan > lysozyme.This study showed the films enriched with chitosan, lysozyme and oregano oil have potential to be used as active biodegradable films with strong antimicrobial effects.

  20. Temperature dependence of lysozyme hydration and the role of elastic energy (United States)

    Wang, Hai-Jing; Kleinhammes, Alfred; Tang, Pei; Xu, Yan; Wu, Yue


    Water plays a critical role in protein dynamics and functions. However, the most basic property of hydration—the water sorption isotherm—remains inadequately understood. Surface adsorption is the commonly adopted picture of hydration. Since it does not account for changes in the conformational entropy of proteins, it is difficult to explain why protein dynamics and activity change upon hydration. The solution picture of hydration provides an alternative approach to describe the thermodynamics of hydration. Here, the flexibility of proteins could influence the hydration level through the change of elastic energy upon hydration. Using nuclear magnetic resonance to measure the isotherms of lysozyme in situ between 18 and 2 °C, the present work provides evidence that the part of water uptake associated with the onset of protein function is significantly reduced below 8 °C. Quantitative analysis shows that such reduction is directly related to the reduction of protein flexibility and enhanced cost in elastic energy upon hydration at lower temperature. The elastic property derived from the water isotherm agrees with direct mechanical measurements, providing independent support for the solution model. This result also implies that water adsorption at charged and polar groups occurring at low vapor pressure, which is known for softening the protein, is crucial for the later stage of water uptake, leading to the activation of protein dynamics. The present work sheds light on the mutual influence of protein flexibility and hydration, providing the basis for understanding the role of hydration on protein dynamics.

  1. Inorganic and Protein Crystal Assembly in Solutions (United States)

    Chernov, A. A.


    The basic kinetic and thermodynamic concepts of crystal growth will be revisited in view of recent AFM and interferometric findings. These concepts are as follows: 1) The Kossel crystal model that allows only one kink type on the crystal surface. The modern theory is developed overwhelmingly for the Kessel model; 2) Presumption that intensive step fluctuations maintain kink density sufficiently high to allow applicability of Gibbs-Thomson law; 3) Common experience that unlimited step bunching (morphological instability) during layer growth from solutions and supercooled melts always takes place if the step flow direction coincides with that of the fluid.

  2. Beneficial effects of increased lysozyme levels in Alzheimer's disease modelled in Drosophila melanogaster. (United States)

    Sandin, Linnea; Bergkvist, Liza; Nath, Sangeeta; Kielkopf, Claudia; Janefjord, Camilla; Helmfors, Linda; Zetterberg, Henrik; Blennow, Kaj; Li, Hongyun; Nilsberth, Camilla; Garner, Brett; Brorsson, Ann-Christin; Kågedal, Katarina


    Genetic polymorphisms of immune genes that associate with higher risk to develop Alzheimer's disease (AD) have led to an increased research interest on the involvement of the immune system in AD pathogenesis. A link between amyloid pathology and immune gene expression was suggested in a genome-wide gene expression study of transgenic amyloid mouse models. In this study, the gene expression of lysozyme, a major player in the innate immune system, was found to be increased in a comparable pattern as the amyloid pathology developed in transgenic mouse models of AD. A similar pattern was seen at protein levels of lysozyme in human AD brain and CSF, but this lysozyme pattern was not seen in a tau transgenic mouse model. Lysozyme was demonstrated to be beneficial for different Drosophila melanogaster models of AD. In flies that expressed Aβ1-42 or AβPP together with BACE1 in the eyes, the rough eye phenotype indicative of toxicity was completely rescued by coexpression of lysozyme. In Drosophila flies bearing the Aβ1-42 variant with the Arctic gene mutation, lysozyme increased the fly survival and decreased locomotor dysfunction dose dependently. An interaction between lysozyme and Aβ1-42 in the Drosophila eye was discovered. We propose that the increased levels of lysozyme, seen in mouse models of AD and in human AD cases, were triggered by Aβ1-42 and caused a beneficial effect by binding of lysozyme to toxic species of Aβ1-42 , which prevented these from exerting their toxic effects. These results emphasize the possibility of lysozyme as biomarker and therapeutic target for AD. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  3. Impact of lysozyme on stability mechanism of nanozirconia aqueous suspension

    Energy Technology Data Exchange (ETDEWEB)

    Szewczuk-Karpisz, Katarzyna, E-mail:; Wiśniewska, Małgorzata


    Highlights: • Adsorption and stabilization-destabilization properties of lysozyme (LSZ) in the nanozirconia-biopolymer solution system were determined. • The stability measurements were performed using turbidimetric method. • Lysozyme macromolecules undergo adsorption on the ZrO{sub 2} surface under electrostatic adsorbent-adsorbate attraction, i.e. at pH 6 and 9. • The biopolymer adsorption impact on the zirconia stability varies at different pH values. - Abstract: The effect of lysozyme (LSZ) presence on the zirconium(IV) oxide (ZrO{sub 2}) aqueous suspension stability was examined. The applied zirconia contains mesopores (with a diameter about 30 nm) and its mean particle size is about 100 nm. To determine the stability mechanism of ZrO{sub 2} suspension in the biopolymer presence, the adsorption and electrokinetic (surface charge density and zeta potential) measurements were performed in the pH range 3–10. The lysozyme adsorption on the nanozirconia surface proceeds mainly through electrostatic forces. Under solid-polymer repulsion conditions, there is no adsorption of lysozyme (pH < 6, C{sub NaCl} 0.01 mol/dm{sup 3}). The increase of solution ionic strength to 0.2 mol/dm{sup 3} causes screening of unfavourable forces and biopolymer adsorption becomes possible. The LSZ addition to the ZrO{sub 2} suspension influences its stability. At pH 3, 4.6 and 7.6, slight improvement of the system stability was obtained. In turn, at pH 9 considerable destabilization of nanozirconia particles covered by polymeric layers occurs.

  4. Probing interactions and phase separations of proteins, colloids, and polymers with light scattering (United States)

    Parmar, Avanish Singh

    The broad objective of my research is to investigate the physical characteristics and interactions of macromolecules and nanoparticles, and the corresponding effects on their phase separation behavior using static and dynamic light scattering (SLS & DLS). Light scattering provides a non-invasive technique for monitoring the in-situ behavior of solutes in solution, including solute interactions, sizes, shapes, aggregation kinetics and even rheological properties of condensed phases. Initially, we investigated lysozyme solutions for the presence of preformed aggregates and clusters that can distort the kinetics of protein crystal nucleation studies in this important model system for protein crystallization. We found that both undersaturated and supersaturated lysozyme solutions contained population of large, pre-existing protein aggregate. Separating these clusters and analyzing their composition with gel chromatography indicated that these clusters represented pre-formed lysozyme aggregates, and not extrinsic protein contamination. We investigated the effect of chaotropic versus kosmotropic ions (water structure breakers vs. structure makers) on the hydration layer and hydrodynamic interactions of hen egg white lysozyme. Surprisingly, neither chaotropic nor kosmotropic ions affected the protein hydration layer. Salt-effects on direct and hydrodynamic protein interactions were determined as function of the solutions ionic strength and temperature. Using both static and dynamic light scattering, we investigated the nucleation of gold nanoparticles forming from supersaturated gold sols. We observed that two well separated populations of nuclei formed essentially simultaneously, with sizes of 3nm vs. several tens of nanometer, respectively. We explore the use of lysozyme as tracer particle for diffusion-base measurements of electrolyte solutions. We showed that the unusual stability of lysozyme and its enhanced colloidal stability enable viscosity measurement of salts

  5. Structure and stability of self-assembled actin-lysozyme complexes in salty water. (United States)

    Sanders, Lori K; Guáqueta, Camilo; Angelini, Thomas E; Lee, Jae-Wook; Slimmer, Scott C; Luijten, Erik; Wong, Gerard C L


    Interactions between actin, an anionic polyelectrolyte, and lysozyme, a cationic globular protein, have been examined using a combination of synchrotron small-angle x-ray scattering and molecular dynamics simulations. Lysozyme initially bridges pairs of actin filaments, which relax into hexagonally coordinated columnar complexes comprised of actin held together by incommensurate one-dimensional close-packed arrays of lysozyme macroions. These complexes are found to be stable even in the presence of significant concentrations of monovalent salt, which is quantitatively explained from a redistribution of salt between the condensed and the aqueous phases.

  6. How do trehalose, maltose and sucrose influence some structural and dynamical properties of lysozyme ? An insight from Molecular Dynamics simulations

    CERN Document Server

    Lerbret, A; Affouard, F; Hedoux, A; Guinet, Y; Descamps, M


    The influence of three well-known disaccharides, namely trehalose, maltose and sucrose, on some structural and dynamical properties of lysozyme has been investigated by means of molecular dynamics computer simulations in the 37-60 wt % concentration range. The effects of sugars on the protein conformation are found relatively weak, in agreement with the preferential hydration of lysozyme. Conversely, sugars seem to increase significantly the relaxation times of the protein. These effects are shown to be correlated to the fractional solvent accessibilities of lysozyme residues and further support the slaving of protein dynamics. Moreover, a significant increase in the relaxation times of lysozyme, sugars and water molecules is observed within the studied concentration range and may result from the percolation of the hydrogen-bond network of sugar molecules. This percolation appears to be of primary importance to explain the influence of sugars on the dynamical properties of lysozyme and water.

  7. The effect of mucine, IgA, urea, and lysozyme on the corrosion behavior of various non-precious dental alloys and pure titanium in artificial saliva. (United States)

    Bilhan, H; Bilgin, T; Cakir, A F; Yuksel, B; Von Fraunhofer, J A


    The corrosion of dental alloys has biological, functional, and aesthetic consequences. Various studies have shown that protein solutions can inhibit the corrosion of alloys. This study is planned to determine the relationship of organic constituents of saliva and the corrosion of dental alloys. The organic constituents are IgA, mucine, urea, and lysozyme which are encountered in the highest amounts in saliva and the dental materials are titanium (Ti), Co-Cr-Mo and Ni-Cr-Mo alloys, and dental amalgam, the most often used metallic components in dentistry. In particular, the interactions between the commonest salivary proteins, IgA, mucine, urea and lysozyme, and Ti, Co-Cr-Mo, Ni-Cr-Mo and dental amalgam were investigated. Each alloy was evaluated by cyclic polarization in each medium. The general anodic and cathodic behavior during forward and reverse cycles, the corrosion and passivation current densities (muA/cm2 ), and the corrosion and the pitting potentials (mV) were determined. The results have shown that Ni-Cr-Mo and dental amalgam alloys are highly susceptible to corrosion in all the investigated media. The Co-Cr-Mo alloy has shown high passive current densities in the solution of mucine and lysozyme in artificial saliva. Titanium instead, has shown a high resistance to corrosion and a stable passive behavior in all media, especially in a solution of mucine and IgA in synthetic saliva. Mucine and IgA, as well as urea and lysozyme, appeared to enhance the formation of a passive film layer on the Ti metal surface, thus inhibiting the corrosion. Based on the study findings, and especially considering the problem of nickel allergy and toxicity of mercury released from dental amalgam, the use of Co-Cr-Mo alloys and Ti to Ni-Cr-Mo alloys is recommended and alternatives to dental amalgam should be sought for patients with impaired salivary flow.

  8. An ignored variable: solution preparation temperature in protein crystallization. (United States)

    Chen, Rui-Qing; Lu, Qin-Qin; Cheng, Qing-Di; Ao, Liang-Bo; Zhang, Chen-Yan; Hou, Hai; Liu, Yong-Ming; Li, Da-Wei; Yin, Da-Chuan


    Protein crystallization is affected by many parameters, among which certain parameters have not been well controlled. The temperature at which the protein and precipitant solutions are mixed (i.e., the ambient temperature during mixing) is such a parameter that is typically not well controlled and is often ignored. In this paper, we show that this temperature can influence protein crystallization. The experimental results showed that both higher and lower mixing temperatures can enhance the success of crystallization, which follows a parabolic curve with an increasing ambient temperature. This work illustrates that the crystallization solution preparation temperature is also an important parameter for protein crystallization. Uncontrolled or poorly controlled room temperature may yield poor reproducibility in protein crystallization.

  9. Phase diagrams and kinetics of phase transitions in protein solutions. (United States)

    Vekilov, Peter G


    The phase behavior of proteins is of interest for fundamental and practical reasons. The nucleation of new phases is one of the last major unresolved problems of nature. The formation of protein condensed phases (crystals, polymers, and other solid aggregates, as well as dense liquids and gels) underlies pathological conditions, plays a crucial role in the biological function of the respective protein, or is an essential part of laboratory and industrial processes. In this review, we focus on phase transitions of proteins in their properly folded state. We first summarize the recently acquired understanding of physical processes underlying the phase diagrams of the protein solutions and the thermodynamics of protein phase transitions. Then we review recent findings on the kinetics of nucleation of dense liquid droplets and crystals. We explore the transition from nucleation to spinodal decomposition for liquid-liquid separation and introduce the new concept of solution-to-crystal spinodal. We review the two-step mechanism of protein crystal nucleation, in which mesoscopic metastable protein clusters serve as precursors to the ordered crystal nuclei. The concepts and mechanisms reviewed here provide powerful tools for control of the nucleation process by varying the solution thermodynamic parameters.

  10. An integrated process for purification of lysozyme, ovalbumin, and ovomucoid from hen egg white. (United States)

    Roy, Ipsita; Rao, M V S; Gupta, Munishwar N


    This article describes an integrated process for simultaneous purification of lysozyme, ovalbumin, and ovomucoid from hen egg white. The crude egg white extract was passed through a cation exchanger Streamline trade mark SP and the bound lysozyme was eluted with 5% ammonium carbonate, pH 9.0, containing 1 M NaCl after elution of avidin. This partially purified lysozyme was further purified 639-fold on dye-linked cellulose beads. Ovalbumin and ovomucoid did not bind to Streamline SP. Ovalbumin could be precipitated from this unbound fraction by 5% trichloroacetic acid, and ovomucoid was removed from the supernatant by precipitation with ethanol. The yields of lysozyme, ovomucoid, and ovalbumin were 77, 94, and 98%, respectively. All the purified proteins showed single bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. All the steps are easily scalable, and the process described here can be used for large-scale simultaneous purification of these proteins in the pure form.

  11. Forced Desorption of Bovine Serum Albumin and Lysozyme from Graphite: Insights from Molecular Dynamics Simulation. (United States)

    Mücksch, Christian; Urbassek, Herbert M


    We use molecular dynamics (MD) simulation to study the adsorption and desorption of two widely different proteins, bovine serum albumin (BSA) and lysozyme, on a graphite surface. The adsorption is modeled using accelerated MD to allow the proteins to find optimum conformations on the surface. Our results demonstrate that the "hard protein" lysozyme retains much of its secondary structure during adsorption, whereas BSA loses it almost completely. BSA has a considerably larger adsorption energy compared to that of lysozyme, which does not scale with chain length. Desorption simulations are carried out using classical steered MD. The BSA molecule becomes fully unzipped during pull-off, whereas several helices survive this process in lysozyme. The unzipping process shows up in the force-distance curve of BSA as a series of peaks, whereas only a single or few, depending on protein orientation, force peaks occur for lysozyme. The maximum desorption force is larger for BSA than for lysozyme, but only by a factor of about 2.3.

  12. Receptor-mediated endocytosis of lysozyme in renal proximal tubules of the frog Rana temporaria

    Directory of Open Access Journals (Sweden)

    E.V. Seliverstova


    Full Text Available The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endosomes, and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intracellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals.

  13. Aggregation of silica nanoparticles directed by adsorption of lysozyme. (United States)

    Bharti, Bhuvnesh; Meissner, Jens; Findenegg, Gerhard H


    The interaction of the globular protein lysozyme with silica nanoparticles of diameter 20 nm was studied in a pH range between the isoelectric points (IEPs) of silica and the protein (pH 3-11). The adsorption affinity and capacity of lysozyme on the silica particles is increasing progressively with pH, and the adsorbed protein induces bridging aggregation of the silica particles. Structural properties of the aggregates were studied as a function of pH at a fixed protein-to-silica concentration ratio which corresponds to a surface concentration of protein well below a complete monolayer in the complete-binding regime at pH > 6. Sedimentation studies indicate the presence of compact aggregates at pH 4-6 and a loose flocculated network at pH 7-9, followed by a sharp decrease of aggregate size near the IEP of lysozyme. The structure of the bridged silica aggregates was studied by cryo-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering. The structure factor S(q) derived from the scattering profiles displays characteristic features of particles interacting by a short-range attractive potential and can be represented by the square-well Percus-Yevick potential model, with a potential depth not exceeding 3k(B)T.

  14. The Lysozyme from Insect (Manduca sexta) is a Cold-Adapted Enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Sotelo-Mundo,R.; Lopez-Zavala, A.; Garcia-Orozco, K.; Arvizu-Flores, A.; Velazquez-Contreras, E.; Valenzuela-Soto, E.; Rojo-Dominguez, A.; Kanost, M.


    Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of {alpha}-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 {sup o}C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.

  15. Terahertz absorption spectroscopy of protein-containing reverse micellar solution (United States)

    Murakami, H.; Toyota, Y.; Nishi, T.; Nashima, S.


    Terahertz time-domain spectroscopy has been carried out for AOT/isooctane reverse micellar solution with myoglobin at the water-to-surfactant molar ratios ( w0) of 0.2 and 4.4. The amplitude of the absorption spectrum increases with increasing the protein concentration at w0 = 0.2, whereas it decreases at w0 = 4.4. The molar extinction coefficients of the protein-filled reverse micelle, and the constituents, i.e., myoglobin, water, and AOT, have been derived by use of the structural parameters of the micellar solution. The experimental results are interpreted in terms of hydration onto the protein and surfactant in the reverse micelle.

  16. Isolation and characterization of a c-type lysozyme from the nurse shark. (United States)

    Hinds Vaughan, Nichole; Smith, Sylvia L


    Lysozyme is a ubiquitous antibacterial enzyme that occurs in numerous invertebrate and vertebrate species. Three forms have been described c-type, g-type and i-type which differ in primary structure. Shark lysozyme has not been characterized; here we report on the isolation and characterization of lysozyme from unstimulated shark (Ginglymostoma cirratum) leukocytes and provide amino acid sequence data across the highly conserved active site of the molecule identifying it to be a c-type lysozyme. A leukocyte lysate was applied either (a) to the first of two sequential DE-52 cellulose columns or alternatively, (b) to a DEAE-Sepharose column. Lysozyme activity in lysate and active fractions was identified by zones of lysis of Micrococcus lysodeikticus cell walls on lysoplates and zones of growth inhibition in agar diffusion assays using Planococcus citreus as the target organism. SDS-PAGE analysis revealed a 14 kDa protein which was identified as lysozyme by mass spectroscopic analysis of peptides, reactivity against anti-HEWL antibodies on a Western blot, hydrolysis of M. lysodeikticus cell walls, and inhibition of growth of P. citreus on AU-gel blots in which the area of growth inhibition correlated to a 14 kDa protein.

  17. Salivary lysozyme in smoking alcohol dependent persons. (United States)

    Waszkiewicz, Napoleon; Zalewska-Szajda, Beata; Zalewska, Anna; Waszkiewicz, Magdalena; Szajda, Slawomir Dariusz; Repka, Bernadeta; Szulc, Agata; Kepka, Alina; Minarowska, Alina; Ladny, Jerzy Robert; Zwierz, Krzysztof


    The purpose of this study was to evaluate the effect of chronic alcohol intoxication and smoking on the concentration and output of salivary lysozyme. Thirty seven men participated in the study, including 17 male smoking alcohol-dependent patients after chronic alcohol intoxication (AS), and 20 control non-smoking male social drinkers (CNS) with no history of alcohol abuse or smoking. The level of lysozyme was assessed by the radial immunodiffusion method. Significantly lower lysozyme output in the AS group compared to the CNS group was found. Moreover, gingival index was significantly higher in AS than in the CNS group. It appeared that the reduced salivary lysozyme output was more likely the result of ethanol action than smoking. In conclusion, persons addicted to alcohol and nicotine have a poorer periodontal status than non-smoking social drinkers, which may partially be due to the diminished protective effects of lysozyme present in the saliva.

  18. Measurements of Protein-Protein Interactions by Size Exclusion Chromatography


    Bloustine, J.; Berejnov, V.; Fraden, S


    A method is presented for determining second virial coefficients (B2) of protein solutions from retention time measurements in size exclusion chromatography. We determine B2 by analyzing the concentration dependence of the chromatographic partition coefficient. We show the ability of this method to track the evolution of B2 from positive to negative values in lysozyme and bovine serum albumin solutions. Our size exclusion chromatography results agree quantitatively with data obtained by light...

  19. Evaluation of alternatives for human lysozyme purification from transgenic rice: impact of phytic acid and buffer. (United States)

    Wilken, Lisa R; Nikolov, Zivko L


    Producing economically competitive recombinant human lysozyme from transgenic rice demands an inexpensive purification process for nonpharmaceutical applications. Human lysozyme is a basic protein, and thus, cation exchange chromatography was the selected method for lysozyme purification. Similar to other protein production systems, the identification of critical impurities in the rice extract was important for the development of an efficient purification process. Previous adsorption data indicated that phytic acid was probably responsible for an unacceptably low cation exchange adsorption capacity. In this study, we confirm that reducing phytic acid concentration improves lysozyme binding capacity and investigate alternative process conditions that reduce phytic acid interference. Compared with the previous best process, the adsorption capacity of human lysozyme was increased from 8.6 to 19.7 mg/mL when rice extract was treated with phytase to degrade phytic acid. Using tris buffer to adjust pH 4.5 extract to pH 6 before adsorption reduced phytic acid interference by minimizing phytic acid-lysozyme interactions, eliminated the need for phytase treatment, and increased the binding capacity to 25 mg/mL. Another method of reducing phytic acid concentration was to extract human lysozyme from rice flour at pH 10 with 50 mM NaCl in 50 mM sodium carbonate buffer. A similar binding capacity (25.5 mg/mL) was achieved from pH 10 extract that was clarified by acidic precipitation and adjusted to pH 6 for adsorption. Lysozyme purities ranged from 95 to 98% for all three processing methods. The tris-mediated purification was the most efficient of the alternatives considered.

  20. SANS and UV-vis spectroscopy studies of resultant structure from lysozyme adsorption on silica nanoparticles. (United States)

    Kumar, Sugam; Aswal, Vinod K; Kohlbrecher, Joachim


    The interaction of lysozyme protein (M.W. 14.7 kD) with two sizes of silica nanoparticles (16 and 25 nm) has been examined in aqueous solution using UV-vis spectroscopy and small-angle neutron scattering (SANS). The measurements were performed on fixed concentration (1 wt %) of nanoparticles and varying concentration of protein in the range 0 to 2 wt %. The adsorption isotherm as obtained using UV-vis spectroscopy suggests strong interaction of the two components and shows an exponential behavior. The saturation values of adsorption are found to be around 90 and 270 protein molecules per particle for 16 and 25 nm sized nanoparticles, respectively. The adsorption of protein on nanoparticles leads to the aggregation of particles and these structures have been studied by SANS. The aggregates are characterized by fractal structure coexisting with unaggregated particles at low protein concentrations and free proteins at higher protein concentrations. Further, contrast variation SANS measurements have been carried out to differentiate the adsorbed and free protein in these systems.

  1. Expression of recombinant human lysozyme in transgenic chicken promotes the growth of Bifidobacterium in the intestine and improves postnatal growth of chicken


    Wang, Hai; Wu, Hongping; Wang, Kejun; Cao, Zhichen; Yu, Kun; Lian, Ling; Lian, Zhengxing


    Lysozyme is one kind of antimicrobial proteins and often used as feed additive which can defend against pathogenic bacteria and enhance immune function of animals. In this study, we have injected the lentiviral vector expressing recombinant human lysozyme (rhLZ) gene into the blastoderm of chicken embryo to investigate the effect of recombinant human lysozyme on postnatal intestinal microbiota distribution and growth performance of chicken. Successfully, we generated 194 transgenic chickens i...

  2. Model-free methods of analyzing domain motions in proteins from simulation : A comparison of normal mode analysis and molecular dynamics simulation of lysozyme

    NARCIS (Netherlands)

    Hayward, S; Kitao, A; Berendsen, HJC


    Model-free methods are introduced to determine quantities pertaining to protein domain motions from normal mode analyses and molecular dynamics simulations, For the normal mode analysis, the methods are based on the assumption that in low frequency modes, domain motions can be well approximated by m

  3. In vivo and in vitro release of lysozyme from cross-linked gelatin hydrogels : a model system for the delivery of antibacterial proteins from prosthetic heart valves

    NARCIS (Netherlands)

    Kuijpers, AJ; van Wachem, PB; van Luyn, MJA; Engbers, GHM; Krijgsveld, J; Zaat, SAJ; Feijen, J


    Prosthetic valve endocarditis may be reduced by the local delivery of antibacterial proteins from the Dacron sewing ring of a prosthetic heart valve. Dacron discs were treated with a carbon dioxide gas plasma to improve the hydrophilicity and thereby enabling homogeneous impregnation with gelatin ty

  4. Laser ablation dynamics and production of thin films of lysozyme

    DEFF Research Database (Denmark)

    Schou, Jørgen; Canulescu, Stela; Matei, Andreea;

    at the Technical University of Denmark (DTU) produced thin films of average thickness up to 300 nm, which not only contained a significant amount of intact molecules, but also maintained the bioactivity. These films were produced by a nanosecond laser in the UV regime at 355 nm with 2 J/cm2. The surprising fact......, there was a considerable ablation weight loss of lysozyme from each shot. This is the first time the ablation by fs-lasers of a protein has been recorded quantitatively. Films of lysozyme produced by fs-laser irradiation will be analysed by MALDI in order to explore if there also is a significant amount of intact...... molecules in the films for fs-laser deposition....

  5. A zinc complex of heparan sulfate destabilises lysozyme and alters its conformation

    Energy Technology Data Exchange (ETDEWEB)

    Hughes, Ashley J. [Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB (United Kingdom); Diamond Light Source Ltd., Diamond House, Didcot, Oxfordshire OX11 0DE (United Kingdom); Hussain, Rohanah [Diamond Light Source Ltd., Diamond House, Didcot, Oxfordshire OX11 0DE (United Kingdom); Cosentino, Cesare; Guerrini, Marco [Istituto di Chimica e Biochimica ' G. Ronzoni' , Via G. Colombo 81, Milano 20133 (Italy); Siligardi, Giuliano [Diamond Light Source Ltd., Diamond House, Didcot, Oxfordshire OX11 0DE (United Kingdom); Yates, Edwin A., E-mail: [Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB (United Kingdom); Rudd, Timothy R., E-mail: [Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB (United Kingdom); Istituto di Chimica e Biochimica ' G. Ronzoni' , Via G. Colombo 81, Milano 20133 (Italy)


    Highlights: Black-Right-Pointing-Pointer Zinc-heparan sulfate complex destabilises lysozyme, a model amyloid protein. Black-Right-Pointing-Pointer Addition of zinc, without heparan sulfate, stabilises lysozyme. Black-Right-Pointing-Pointer Heparan sulfate cation complexes provide alternative protein folding routes. -- Abstract: The naturally occurring anionic cell surface polysaccharide heparan sulfate is involved in key biological activities and is implicated in amyloid formation. Following addition of Zn-heparan sulfate, hen lysozyme, a model amyloid forming protein, resembled {beta}-rich amyloid by far UV circular dichroism (increased {beta}-sheet: +25%), with a significantly reduced melting temperature (from 68 to 58 Degree-Sign C) by fluorescence shift assay. Secondary structure stability of the Zn-heparan sulfate complex with lysozyme was also distinct from that with heparan sulfate, under stronger denaturation conditions using synchrotron radiation circular dichroism. Changing the cation associated with heparan sulfate is sufficient to alter the conformation and stability of complexes formed between heparan sulfate and lysozyme, substantially reducing the stability of the protein. Complexes of heparan sulfate and cations, such as Zn, which are abundant in the brain, may provide alternative folding routes for proteins.

  6. Comparative insight into surfactants mediated amyloidogenesis of lysozyme. (United States)

    Chaturvedi, Sumit K; Khan, Javed M; Siddiqi, Mohammad K; Alam, Parvez; Khan, Rizwan H


    Electrostatic and hydrophobic interactions have an important role in the protein aggregation. In this study, we have investigated the effect of charge and hydrophobicity of oppositely charged surfactants i.e., anionic (AOT and SDS) and cationic (CTAB and DTAB) on hen egg white lysozyme at pH 9.0 and 13.0, respectively. We have employed various methods such as turbidity measurements, Rayleigh light scattering, ThT, Congo red and ANS dye binding assays, far-UV CD, atomic force microscopy, transmission electron and fluorescence microscopy. At lower molar ratio, both anionic and cationic surfactants promote amyloid fibril formation in lysozyme at pH 9.0 and 13.0, respectively. The aggregation was proportionally increased with respect to protein concentration and hydrophobicity of surfactant. The morphology of aggregates at both the pH was fibrillar in structure, as visualized by dye binding and microscopic imaging techniques. Initially, the interaction between surfactants and lysozyme was electrostatic and then hydrophobic as investigated by ITC. This study demonstrates the crucial role of charge and hydrophobicity during amyloid fibril formation.

  7. pH dependence of the kinetics of interfacial tension changes during protein adsorption from sessile droplets on FEP-Teflon

    NARCIS (Netherlands)

    VanderVegt, W; Norde, W; VanderMei, HC; Busscher, HJ

    Interfacial tension changes during protein adsorption at both the solid-liquid and the liquid-vapor interface were measured simultaneously by ADSA-P from sessile droplets of protein solutions on fluoroethylenepropylene-Teflon. Four globular proteins of similar size, viz. lysozyme, ribonuclease,

  8. Refolding of detergent-denatured lysozyme using β-cyclodextrin-assisted ion exchange chromatography. (United States)

    Zhang, Li; Zhang, Qinming; Wang, Chaozhan


    Chromatography-based protein refolding is widely used. Detergent is increasingly used for protein solubilization from inclusion bodies. Therefore, it is necessary to develop a refolding method for detergent-denatured/solubilized proteins based on liquid chromatography. In the present work, sarkosyl-denatured/dithiothreitol-reduced lysozyme was used as a model, and a refolding method based on ion exchange chromatography, assisted by β-cyclodextrin, was developed for refolding detergent-denatured proteins. Many factors affecting the refolding, such as concentration of urea, concentration of β-cyclodextrin, pH and flow rate of mobile phases, were investigated to optimize the refolding conditions for sarkosyl-denatured lysozymes. The results showed that the sarkosyl-denatured lysozyme could be successfully refolded using β-cyclodextrin-assisted ion exchange chromatography.

  9. Molecular cloning, sequence analysis and phylogeny of first caudata g-type lysozyme in axolotl (Ambystoma mexicanum). (United States)

    Yu, Haining; Gao, Jiuxiang; Lu, Yiling; Guang, Huijuan; Cai, Shasha; Zhang, Songyan; Wang, Yipeng


    Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate.

  10. Effect of Fe{sub 3}O{sub 4} magnetic nanoparticles on lysozyme amyloid aggregation

    Energy Technology Data Exchange (ETDEWEB)

    Bellova, Andrea; Koneracka, Martina; Kopcansky, Peter; Tomasovicova, Natalia; Timko, Milan; Bagelova, Jaroslava; Gazova, Zuzana [Department of Biophysics, Department of Magnetism, Institute of Experimental Physics, Slovak Academy of Science, Watsonova 47, 04001 Kosice (Slovakia); Bystrenova, Eva; Valle, Francesco; Biscarini, Fabio, E-mail: [CNR-Instituto per lo Studio dei Materiali Nanostrutturati, via Gobetti 101, I-40129 Bologna (Italy)


    Peptide amyloid aggregation is a hallmark of several human pathologies termed amyloid diseases. We have investigated the effect of electrostatically stabilized magnetic nanoparticles of Fe{sub 3}O{sub 4} on the amyloid aggregation of lysozyme, as a prototypical amyloidogenic protein. Thioflavin T fluorescence assay and atomic force microscopy were used for monitoring the inhibiting and disassembly activity of magnetic nanoparticles of Fe{sub 3}O{sub 4}. We have found that magnetic Fe{sub 3}O{sub 4} nanoparticles are able to interact with lysozyme amyloids in vitro leading to a reduction of the amyloid aggregates, thus promoting depolymerization; the studied nanoparticles also inhibit lysozyme amyloid aggregation. The ability to inhibit lysozyme amyloid formation and promote lysozyme amyloid disassembly exhibit concentration-dependent characteristics with IC50 = 0.65 mg ml{sup -1} and DC50 = 0.16 mg ml{sup -1} indicating that nanoparticles interfere with lysozyme aggregation already at stoichiometric concentrations. These features make Fe{sub 3}O{sub 4} nanoparticles of potential interest as therapeutic agents against amyloid diseases and their non-risk exploitation in nanomedicine and nanodiagnostics.

  11. Radiation effects on viscosimetry of protein based solutions (United States)

    Sabato, S. F.; Lacroix, M.


    Due to their good functional properties allied to their excellent nutritional value, milk protein isolates and soy protein concentrates have gained a crescent interest. These proteins could have their structural properties improved when some treatments are applied, such as gamma irradiation, alone or in presence of other compounds, as a plasticizer. In this work, solutions of those proteins were mixed with a generally recognized as safe plasticizer, glycerol. These mixtures (8% protein (w/v) base) at two ratios 1:1 and 2:1 (protein:glycerol) were submitted to a gamma irradiation treatment ( 60Co), at doses 0, 5, 15 and 25 kGy, and their rheological performance was studied. As irradiation dose increased viscosity measurements decayed significantly ( pwhey/glycerol remained almost constant as irradiation dose increases. In the case of soy protein isolate and sodium caseinate, a mixture of 2:1 showed a significant higher viscosity ( p<0.05) than a mixture of 1:1.

  12. Soliton solutions for Davydov solitons in α-helix proteins (United States)

    Taghizadeh, N.; Zhou, Qin; Ekici, M.; Mirzazadeh, M.


    The propagation equation for describing Davydov solitons in α-helix proteins has been investigated analytically. There are seven integration tools to extract analytical soliton solutions. They are the Ricatti equation expansion approach, ansatz scheme, improved extended tanh-equation method, the extend exp(-Ψ(τ)) -expansion method, the extended Jacobi elliptic function expansion method, the extended trial equation method and the extended G ' / G - expansion method.

  13. Studies on the Refolding of Egg White Lysozyme Denatured by Urea Using "Phase Diagram" Method of Fluorescence

    Institute of Scientific and Technical Information of China (English)

    BIAN Liu-Jiao; DONG Fa-Xin; LIANG Chang-Li; YANG Xiao-Yan; LIU Li


    The refolding of reduced and non-reducing egg white lysozymes in a urea solution was studied by a "phase diagram" method of fluorescence.The result showed that in the refolding of the reduced egg white lysozyme,an intermediate state of an egg white lysozyme exists at the urea concentrations in a final renaturation solution being about 4.5 mol/L,their refolding follows a three-state model; while in the refolding of the non-reducing egg white iysozyme,two intermediate states exist at the urea concentrations being separately 4.0 and 2.5 mol/L,and their refolding follows a four-state model.Through the comparison between the unfolding and refolding of an egg white lysozyme in the urea solution,it was found that both of the refolding of reduced and non-reducing egg white lysozyme molecules was irreversible to their unfolding in the urea solution.Finally,a suggested refolding was separately presented for the reduced and non-reducing egg white lysozymes in the urea solution.

  14. Evidence of Conformational Changes in Adsorbed Lysozyme Molecule on Silver Colloids

    CERN Document Server

    Chandra, Goutam; Dasgupta, Swagata; Roy, Anushree


    In this article, we discuss metal-protein interactions in the Ag-lysozyme complex by spectroscopic measurements. The analysis of the variation in relative intensities of SERS bands reveal the orientation and the change in conformation of the protein molecules on the Ag surface with time. The interaction kinetics of metal-protein complexes has been analyzed over a period of three hours via both Raman and absorption measurements. Our analysis indicates that the Ag nanoparticles most likely interact with Trp-123 which is in close proximity to Phe-34 of the lysozyme molecule.

  15. Extraction of lysozyme, alpha-chymotrypsin, and pepsin into reverse micelles formed using an anionic surfactant, isooctane, and water. (United States)

    Chang, Q; Liu, H; Chen, J


    The extraction of lysozyme, alpha-chymotrypsin, and pepsin from buffered salt solutions into reverse micelles was examined at different pH values and surfactant concentrations. The reverse micelles was formed by mixing aqueous buffer supplemented with KCl and an organic phase of isooctane(2,2,4-trimethylpentane), containing the anionic surfactant, Aerosol O. T. (dioctyl ester of sodium sulfosuccinic acid). The technique of dynamic laser scattering was used to measure the size of reverse micelles which were in equilibrium with the aqueous phase. It was found that the size of the reverse micelles decreased with increasing ionic strength but increased with increasing AOT concentration. In the process of extraction, the reverse micelles might have rearranged themselves to host the protein. The sizes of protein-filled and -unfilled reverse micelles were different, and an open equilibrium could be reached between them. Under the extraction conditions, only a small number of micelles were found to contain protein.

  16. Preparation and Preliminary Characterization of Crystallizing Fluorescent Derivatives of Chicken Egg White Lysozyme (United States)

    Sumida, John; Forsythe, Elizabeth L.; Pusey, Marc L.; Whitaker, Ann F. (Technical Monitor)


    Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. While most proteins are intrinsically fluorescent, working at crystallization concentrations require the use of covalently prepared derivatives added as tracers. This approach requires derivatives that do not markedly affect the crystal packing. We have prepared fluorescent derivatives of chicken egg white lysozyme with probes bound to one of two different sites on the protein molecule. Lucifer yellow and 5-(2-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS) have been attached to the side chain carboxyl of Asp(sup 101) using a carbodiimide coupling procedure. Asp(sup 101) lies within the active site cleft, and it is believed that the probes are "buried" within that cleft. Lucifer yellow and MANS probes with iodoacetamide reactive groups have been bound to His(sup 15), located on the "back side" of the molecule relative to the active site. All the derivatives fluoresce in the solution and the crystalline states. Fluorescence characterization has focused on determination of binding effects on the probe quantum yield, lifetime, absorption and emission spectra, and quenching by added solutes. Quenching studies show that, as postulated, the Asp(sup 101)-bound probes are partially sheltered from the bulk solution by their location within the active site cleft. Probes bound to His(sup 15) have quenching constants about equal to those for the free probes, indicating that this site is highly exposed to the bulk solution.

  17. A comparison of the physical properties of ultrasonically synthesized lysozyme- and BSA-shelled microbubbles. (United States)

    Vong, Fiona; Son, Younggyu; Bhuiyan, Sadia; Zhou, Meifang; Cavalieri, Francesca; Ashokkumar, Muthupandian


    Ultrasonic technique has been used for synthesising protein microspheres possessing specific physical and functional properties. Various proteins have been used as shell materials under different experimental conditions. In previous studies, thermal or chemical denaturation of the proteins was used to obtain stable bovine-serum albumin (BSA) and lysozyme microbubbles (MBs), respectively. It is ideal to establish a generic procedure to synthesise microspheres irrespective of the nature of the protein. In order to see if a generic procedure can be established, ultrasonic synthesis of lysozyme and BSA MBs was carried out under similar experimental conditions and their properties were evaluated. The size, size distribution and the stability of the MBs were significantly different for the lysozyme and BSA MBs. The size and size distribution of the lysozyme coated MBs were larger than BSA bubbles. The mechanical strength of MBs against the shear forces, generated when irradiated by high frequency ultrasound, was studied using pulsed-sonoluminescence (SL). This study indicated that lysozyme MBs were significantly more stable than BSA MBs. An increase in mechanical strength of the MBs may lead to an increase in their storage lifetime and stability against gas diffusion. Possible reasons for such observations have been discussed.

  18. On the adsorption of magnetite nanoparticles on lysozyme amyloid fibrils. (United States)

    Majorosova, Jozefina; Petrenko, Viktor I; Siposova, Katarina; Timko, Milan; Tomasovicova, Natalia; Garamus, Vasil M; Koralewski, Marceli; Avdeev, Mikhail V; Leszczynski, Błażej; Jurga, Stefan; Gazova, Zuzana; Hayryan, Shura; Hu, Chin-Kun; Kopcansky, Peter


    An adsorption of magnetic nanoparticles (MNP) from electrostatically stabilized aqueous ferrofluids on amyloid fibrils of hen egg white lysozyme (HEWL) in 2mg/mL acidic dispersions have been detected for the MNP concentration range of 0.01-0.1vol.%. The association of the MNP with amyloid fibrils has been characterized by transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS) and magneto-optical measurements. It has been observed that the extent of adsorption is determined by the MNP concentration. When increasing the MNP concentration the formed aggregates of magnetic particles repeat the general rod-like structure of the fibrils. The effect is not observed when MNP are mixed with the solution of lysozyme monomers. The adsorption has been investigated with the aim to clarify previously found disaggregation activity of MNP in amyloid fibrils dispersions and to get deeper insight into interaction processes between amyloids and MNP. The observed effect is also discussed with respect to potential applications for ordering lysozyme amyloid fibrils in a liquid crystal phase under external magnetic fields.

  19. Controlled protein release from electrospun biodegradable fiber mesh composed of poly(epsilon-caprolactone) and poly(ethylene oxide). (United States)

    Kim, Taek Gyoung; Lee, Doo Sung; Park, Tae Gwan


    A blend mixture of poly(epsilon-caprolactone) (PCL) and poly(ethylene oxide) (PEO) was electrospun to produce fibrous meshes that could release a protein drug in a controlled manner. Various biodegradable polymers, such as poly(l-lactic acid) (PLLA), poly(epsilon-caprolactone) (PCL), and poly(d,l-lactic-co-glycolic acid) (PLGA) were dissolved, along with PEO and lysozyme, in a mixture of chloroform and dimethylsulfoxide (DMSO). The mixture was electrospun to produce lysozyme loaded fibrous meshes. Among the polymers, the PCL/PEO blend meshes showed good morphological stability upon incubation in the buffer solution, resulting in controlled release of lysozyme over an extended period with reduced initial bursts. With varying the PCL/PEO blending ratio, the release rate of lysozyme from the corresponding meshes could be readily modulated. The lysozyme release was facilitated by increasing the amount of PEO, indicating that entrapped lysozyme was mainly released out by controlled dissolution of PEO from the blend meshes. Lysozyme released from the electrospun fibers retained sufficient catalytic activity.

  20. Protein-complex structure completion using IPCAS (Iterative Protein Crystal structure Automatic Solution). (United States)

    Zhang, Weizhe; Zhang, Hongmin; Zhang, Tao; Fan, Haifu; Hao, Quan


    Protein complexes are essential components in many cellular processes. In this study, a procedure to determine the protein-complex structure from a partial molecular-replacement (MR) solution is demonstrated using a direct-method-aided dual-space iterative phasing and model-building program suite, IPCAS (Iterative Protein Crystal structure Automatic Solution). The IPCAS iteration procedure involves (i) real-space model building and refinement, (ii) direct-method-aided reciprocal-space phase refinement and (iii) phase improvement through density modification. The procedure has been tested with four protein complexes, including two previously unknown structures. It was possible to use IPCAS to build the whole complex structure from one or less than one subunit once the molecular-replacement method was able to give a partial solution. In the most challenging case, IPCAS was able to extend to the full length starting from less than 30% of the complex structure, while conventional model-building procedures were unsuccessful.

  1. Effects of solute-solute interactions on protein stability studied using various counterions and dendrimers.

    Directory of Open Access Journals (Sweden)

    Curtiss P Schneider

    Full Text Available Much work has been performed on understanding the effects of additives on protein thermodynamics and degradation kinetics, in particular addressing the Hofmeister series and other broad empirical phenomena. Little attention, however, has been paid to the effect of additive-additive interactions on proteins. Our group and others have recently shown that such interactions can actually govern protein events, such as aggregation. Here we use dendrimers, which have the advantage that both size and surface chemical groups can be changed and therein studied independently. Dendrimers are a relatively new and broad class of materials which have been demonstrated useful in biological and therapeutic applications, such as drug delivery, perturbing amyloid formation, etc. Guanidinium modified dendrimers pose an interesting case given that guanidinium can form multiple attractive hydrogen bonds with either a protein surface or other components in solution, such as hydrogen bond accepting counterions. Here we present a study which shows that the behavior of such macromolecule species (modified PAMAM dendrimers is governed by intra-solvent interactions. Attractive guanidinium-anion interactions seem to cause clustering in solution, which inhibits cooperative binding to the protein surface but at the same time, significantly suppresses nonnative aggregation.

  2. Inhibition of lysozyme amyloidogenesis by phospholipids. Focus on long-chain dimyristoylphosphocholine. (United States)

    Ponikova, Slavomira; Kubackova, Jana; Bednarikova, Zuzana; Marek, Jozef; Demjen, Erna; Antosova, Andrea; Musatov, Andrey; Gazova, Zuzana


    Protein amyloid aggregation is an important pathological feature of a group of different degenerative human diseases called amyloidosis. We tested effect of two phospholipids, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) on amyloid aggregation of hen egg white (HEW) lysozyme in vitro. Effect of phospholipids was investigated using spectroscopic techniques (fluorescence and CD spectroscopy), atomic force microscopy and image analysis. Phospholipids DMPC and DHPC are able dose-dependently inhibit lysozyme fibril formation. The length of the phospholipid tails and different structural arrangement of the phospholipid molecules affect inhibitory activity; long-chain DMPC inhibits fibrillization more efficiently. Interestingly, interference of DMPC with lysozyme amyloid fibrils has no effect on their morphology or amount. Phospholipid molecules have significant effect on lysozyme amyloid fibrillization. We suggest that inhibitory activity is due to the interference of phospholipids with lysozyme leading to the blocking of the intermolecular protein interactions important for formation of the cross-β structure within the core of the fibrils. The higher inhibitory activity of DMPC is probably due to adsorption of protein molecules on the liposome surfaces which caused decrease of species needed for fibrillization. Interaction of the phospholipids with formed fibrils is not sufficient enough to interrupt the bonds in β-sheets which are required for destroying of amyloid fibrils. The obtained results contribute to a better understanding of the effect of phospholipids on amyloid fibrillization of the lysozyme. The data suggest that DMPC and DHPC phospholipids represent agents able to modulate lysozyme amyloid aggregation. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Terahertz mechanical vibrations in lysozyme: Raman spectroscopy vs modal analysis (United States)

    Carpinteri, Alberto; Lacidogna, Giuseppe; Piana, Gianfranco; Bassani, Andrea


    The mechanical behaviour of proteins is receiving an increasing attention from the scientific community. Recently it has been suggested that mechanical vibrations play a crucial role in controlling structural configuration changes (folding) which govern proteins biological function. The mechanism behind protein folding is still not completely understood, and many efforts are being made to investigate this phenomenon. Complex molecular dynamics simulations and sophisticated experimental measurements are conducted to investigate protein dynamics and to perform protein structure predictions; however, these are two related, although quite distinct, approaches. Here we investigate mechanical vibrations of lysozyme by Raman spectroscopy and linear normal mode calculations (modal analysis). The input mechanical parameters to the numerical computations are taken from the literature. We first give an estimate of the order of magnitude of protein vibration frequencies by considering both classical wave mechanics and structural dynamics formulas. Afterwards, we perform modal analyses of some relevant chemical groups and of the full lysozyme protein. The numerical results are compared to experimental data, obtained from both in-house and literature Raman measurements. In particular, the attention is focused on a large peak at 0.84 THz (29.3 cm-1) in the Raman spectrum obtained analyzing a lyophilized powder sample.

  4. Designing and defining dynamic protein cage nanoassemblies in solution. (United States)

    Lai, Yen-Ting; Hura, Greg L; Dyer, Kevin N; Tang, Henry Y H; Tainer, John A; Yeates, Todd O


    Central challenges in the design of large and dynamic macromolecular assemblies for synthetic biology lie in developing effective methods for testing design strategies and their outcomes, including comprehensive assessments of solution behavior. We created and validated an advanced design of a 600-kDa protein homododecamer that self-assembles into a symmetric tetrahedral cage. The monomeric unit is composed of a trimerizing apex-forming domain genetically linked to an edge-forming dimerizing domain. Enhancing the crystallographic results, high-throughput small-angle x-ray scattering (SAXS) comprehensively contrasted our modifications under diverse solution conditions. To generate a phase diagram associating structure and assembly, we developed force plots that measure dissimilarity among multiple SAXS data sets. These new tools, which provided effective feedback on experimental constructs relative to design, have general applicability in analyzing the solution behavior of heterogeneous nanosystems and have been made available as a web-based application. Specifically, our results probed the influence of solution conditions and symmetry on stability and structural adaptability, identifying the dimeric interface as the weak point in the assembly. Force plots comparing SAXS data sets further reveal more complex and controllable behavior in solution than captured by our crystal structures. These methods for objectively and comprehensively comparing SAXS profiles for systems critically affected by solvent conditions and structural heterogeneity provide an enabling technology for advancing the design and bioengineering of nanoscale biological materials.

  5. Molecular Characterization of a Lysozyme Gene and Its Altered Expression Profile in Crowded Beet Webworm (Loxostege sticticalis) (United States)

    Kong, Hailong; Lv, Min; Mao, Nian; Wang, Cheng; Cheng, Yunxia; Zhang, Lei; Jiang, Xingfu; Luo, Lizhi


    There is growing evidence that insects living in high-density populations exhibit an increase in immune function to counter a higher risk of disease. This phenomenon, known as density-dependent prophylaxis, has been experimentally tested in a number of insect species. Although density-dependent prophylaxis is especially prevalent in insects exhibiting density-dependent phase polyphenism, the molecular mechanism remains unclear. Our previous study demonstrated that the antibacterial activity of lysozyme is important for this process in the beet webworm Loxostege sticticalis. In this study, a lysozyme cDNA from L. sticticalis was cloned and characterized. The full-length cDNA is 1078 bp long and contains an open reading frame of 426 bp that encodes 142 amino acids. The deduced protein possesses structural characteristics of a typical c-type lysozyme and clusters with c-type lysozymes from other Lepidoptera. LsLysozyme was found to be expressed throughout all developmental stages, showing the highest level in pupae. LsLysozyme was also highly expressed in the midgut and fat body. Elevated LsLysozyme expression was observed in L. sticticalis larvae infected by Beauveria bassiana and in larvae reared under crowding conditions. In addition, the expression level of LsLysozyme in infected larvae reared at a density of 10 larvae per jar was significantly higher compared to those reared at a density of l or 30 larvae per jar. These results suggest that larval crowding affects the gene expression profile of this lysozyme. This study provides additional insight into the expression of an immune-associated lysozyme gene and helps us to better understand the immune response of L. sticticalis under crowding conditions. PMID:27575006

  6. C-terminus of TRAP in Staphylococcus can enhance the activity of lysozyme and lysostaphin

    Institute of Scientific and Technical Information of China (English)

    Guang Yang; Ningsheng Shao; Yaping Gao; Jiannan Feng; Yong Huang; Shaohua Li; Yu Liu; Chuan Liu; Ming Fan; Beifen Shen


    In Staphylococcus aureus, the target of RNAⅢ activating protein (TRAP) is a membrane-associated protein whose Cterminus can be used as a vaccine to provide protection against staphylococcal infection. Here, we show for the first time by surface plasmon resonance and enzyme-linked immunosorbent assay that TRAP can specifically bind lysozyme and iysostaphin through its C-terminus (amino acids 155-167) and enhance lysozomal activities in vitro. It was also found that the traP mutant strain is more resistant to iysostaphin than wild-type. Our previous data showed that the C-terminus of TRAP might be extracellular. So our results suggested that the C-terminus of TRAP could act as the specific targeting protein of the lysozyme/lysostaphin on the S. aureus cell wall and the biological significance of the interaction might be to facilitate lysozyme/lysostaphin-mediated cell iysis.

  7. Expression, Characterization and Antimicrobial Ability of a Variant T4 Lysozyme in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Ning SUN; Sanfeng CHEN; Xiangming XIE; Yueju WANG; Gangqiang LI; Nan WANG; Dehu LIU


    T4 lysozyme was engineered with disulfide bonds and expressed in Pichia pastoris. The secreted proteins were purified and made into powder by lyophiliza-tion. Recombinant protein purity was more than 70% measured by HPLC. The lytic activity of variant T4-lysozyme was measured by the lysis of the cel wal of Xan-thomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, Ralstonia solanacearum comb. nov, Clavibacter michiganensis subsp. michiganensis, X. campestris pv. mal-vacearum, Fusarium oxysporium sp. vasinfectum, Verticil ium dahliae kleb. Inhibition zone assay showed that variant T4 lysozyme significantly inhibited X. o. oryzicola and X. c. malvacearum. The antifungal activities of this protein against F. o. vasin-fectum and V. d. kleb were also analyzed.

  8. Crystal growth in a three-phase system: diffusion and liquid-liquid phase separation in lysozyme crystal growth. (United States)

    Heijna, M C R; van Enckevort, W J P; Vlieg, E


    In the phase diagram of the protein hen egg-white lysozyme, a region is present in which the lysozyme solution demixes and forms two liquid phases. In situ observations by optical microscopy show that the dense liquid droplets dissolve when crystals grow in this system. During this process the demixed liquid region retracts from the crystal surface. The spatial distribution of the dense phase droplets present special boundary conditions for Fick's second law for diffusion. In combination with the cylindrical symmetry provided by the kinetically roughened crystals, this system allows for a full numerical analysis. Using experimental data for setting the boundary conditions, a quasi-steady-state solution for the time-dependent concentration profile was shown to be valid. Comparison of kinetically rough growth in a phase separated system and in a nonseparated system shows that the growth kinetics for a three-phase system differs from a two-phase system, in that crystals grow more slowly but the duration of growth is prolonged.

  9. Specific delivery of captopril to the kidney with the prodrug captopril-lysozyme

    NARCIS (Netherlands)

    Kok, R.J; Moolenaar, Frits; de Zeeuw, D; Meijer, D.K F

    Low-molecular-weight proteins (LMWPs) accumulate in the proximal tubular cells of the kidney, which makes these proteins interesting tools for renal drug targeting. We studied this approach using the LMWP lysozyme as a carrier for the angiotensin-converting enzyme inhibitor captopril. Captopril was

  10. Synthesis of Ag{sub 2}S nanorods by biomimetic method in the lysozyme matrix

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Dezhi, E-mail:; Zhang, Li; He, Guoxu; Zhang, Qiuxia


    Graphical abstract: - Highlights: • Firstly, Ag{sub 2}S nanorods were synthesized by biomimetic method in the lysozyme solutions. • The study of the interaction between Ag{sup +} and the lysozyme. • Discussion of possible formation mechanism of Ag{sub 2}S nanorods. • The synthesis process of lyso-conjugated Ag{sub 2}S nanocrystals is facile, effective and environment friendly. - Abstract: Ag{sub 2}S nanorods were successfully synthesized by biomimetic route in the lysozyme solution at physiological temperature and atmospheric pressure. The transmission electron microscopy (TEM) images revealed that the prepared nanorods are uniform and monodisperse with homogeneous size about 50 nm in diameter and 150 nm in length. The optical property of Ag{sub 2}S nanocrystals was studied by the ultraviolet–visible (UV–vis) and photoluminescence (PL) spectroscopy, the results show that the products exhibit well-defined emission at 471 nm and 496 nm excited by 292 nm. The interaction of Ag{sup +}/Ag{sub 2}S with the lysozyme was investigated through Fourier transform infrared (FT-IR) spectroscopy, which shows that the cooperation effect of the lysozyme and Ag{sup +} could be responsible for the formation of as obtained Ag{sub 2}S nanorods.

  11. Efficient purification of lysozyme from egg white by 2-mercapto-5-benzimidazolesulfonic acid modified Fe3O4/Au nanoparticles. (United States)

    Zhu, Xinjun; Zhang, Lianying; Fu, Aiyun; Yuan, Hao


    2-Mercapto-5-benzimidazolesulfonic acid (MBISA) modified Fe3O4/Au nanoparticles were synthesized in aqueous solution and characterized by photo correlation spectroscopy (PCS) and vibrating sample magnetometer (VSM). The so-obtained Fe3O4/Au-MBISA nanoparticles were capable of specific adsorbing lysozyme. The maximum amount of lysozyme adsorbed on 1.0mg Fe3O4/Au-MBISA nanoparticles was 346μg. The lysozyme desorption behavior was studied and the lysozyme recovery from Fe3O4/Au-MBISA nanoparticles approached 100% under optimal conditions, and the reusability studies showed that the nanoparticles could maintain about 91% of the initial lysozyme adsorption capacity after 7 repeated adsorption-elution cycles. The Fe3O4/Au-MBISA nanoparticles were used in the purification of lysozyme from chicken egg white, which was verified by a single SDS-PAGE band. Therefore, the obtained Fe3O4/Au-MBISA nanoparticles exhibited excellent performance in the direct purification of lysozyme from egg white.

  12. Determination of protein phase diagrams by microbatch experiments: exploring the influence of precipitants and pH. (United States)

    Baumgartner, Kai; Galm, Lara; Nötzold, Juliane; Sigloch, Heike; Morgenstern, Josefine; Schleining, Kristina; Suhm, Susanna; Oelmeier, Stefan A; Hubbuch, Jürgen


    Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry. Proteins can either be soluble, crystalline or precipitated. Additionally liquid-liquid phase separation, gelation and skin formation can occur. A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed. For lysozyme from chicken egg white, human lysozyme, glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values – pH 3, 5, 7 and 9. Sodium chloride, ammonium sulfate, polyethylene glycol 300 and polyethylene glycol 1000 were used as precipitants. Crystallizing conditions could be found for lysozyme from chicken egg white using sodium chloride, for human lysozyme using sodium chloride or ammonium sulfate and glucose isomerase using ammonium sulfate. PEG caused destabilization of human lysozyme and glucose oxidase solutions or a balance of stabilizing and destabilizing effects for glucose isomerase near the isoelectric point. This work presents a systematic generation and extensive study of phase diagrams of proteins. Thus, it adds to the general understanding of protein behavior in liquid formulation and presents a convenient methodology applicable to any protein solution.

  13. Superheating of ice crystals in antifreeze protein solutions


    Celik, Yeliz; Graham, Laurie A.; Mok, Yee-Foong; Bar, Maya; Davies, Peter L.; Braslavsky, Ido


    It has been argued that for antifreeze proteins (AFPs) to stop ice crystal growth, they must irreversibly bind to the ice surface. Surface-adsorbed AFPs should also prevent ice from melting, but to date this has been demonstrated only in a qualitative manner. Here we present the first quantitative measurements of superheating of ice in AFP solutions. Superheated ice crystals were stable for hours above their equilibrium melting point, and the maximum superheating obtained was 0.44 °C. When me...

  14. A generalized free-solvent model for the osmotic pressure of multi-component solutions containing protein-protein interactions. (United States)

    McBride, Devin W; Rodgers, V G J


    The free-solvent model has been shown to have excellent predictability of the osmotic pressure for single and binary non-interactive proteins in aqueous solutions. Here the free-solvent model is extended to be more generalized by including the contributions of intra- and inter-protein interactions to the osmotic pressure of a solution in the form of homo- and hetero-multimers. The solute-solvent interactions are considered to be unique for each homo- and hetero-multimer in solution. The effect of the various generalized free-solvent model parameters on the osmotic pressure are examined for a single protein solution with a homo-dimer, a binary protein solution with no protein-protein interactions, and a binary protein solution with a hetero-dimer. Finally, the limitations associated with the generalized free-solvent model are discussed.

  15. Chitosan-based membrane chromatography for protein adsorption and separation. (United States)

    Liu, Yezhuo; Feng, Zhicheng; Shao, Zhengzhong; Chen, Xin


    A chitosan-based membrane chromatography was set up by using natural chitosan/carboxymethylchitosan (CS/CMCS) blend membrane as the matrix. The dynamic adsorption property for protein (lysozyme as model protein) was detailed discussed with the change in pore size of the membrane, the flow rate and the initial concentration of the feed solution, and the layer of membrane in membrane stack. The best dynamic adsorption capacity of lysozyme on the CS/CMCS membrane chromatography was found to be 15.3mg/mL under the optimal flow conditions. Moreover, the CS/CMCS membrane chromatography exhibited good repeatability and reusability with the desorption efficiency of ~90%. As an application, lysozyme and ovalbumin were successfully separated from their binary mixture through the CS/CMCS membrane chromatography. This implies that such a natural chitosan-based membrane chromatography may have great potential on the bioseparation field in the future.

  16. 信息动态%Investigation for Relationship Between Heat Treatment Process and Immunogenicity of Lysozyme in Egg By Spectroscopic Method

    Institute of Scientific and Technical Information of China (English)


    Thermal denaturation for lysozyme protein of egg has been investigated by using circular dichroism spectra and fluorescence spectra systematically. The corresponding immunogenicity of lysozyme protein has been tested with ELISA experiments. In addition, bioinformatics methods have also been used to investigate the spectral characteristics, corresponding immunogenicity change and conformational changes of the protein during thermal denaturation. Results from this study should be useful to the establishment of a spectral method examining the extent of thermal denaturation of the protein.

  17. Involvement of PKA, PKC, and Ca2+ in LPS-activated expression of the chicken lysozyme gene. (United States)

    Regenhard, P; Goethe, R; Phi-van, L


    The lysozyme gene is activated in myelomonocytic HD11 cells in response to LPS. In this study, we described the involvement of LPS-activated signal transduction pathways in activation of the lysozyme gene. Pre-treatment of HD 11 cells with H-89, H-7, TMB-8, or KN-93 resulted in inhibition of the LPS-enhanced lysozyme expression, suggesting that PKA, PKC, and Ca2+-dependent protein kinases participate in the LPS activation. CaMKII seems to be required for the processing of lysozyme transcripts. TPA and calcium ionophore A23187, when separately added to HD11 cells, stimulated the lysozyme expression effectively, and forskolin was ineffective. It is interesting that simultaneous treatment of cells with forskolin and calcium ionophore A23187 resulted in a potentiated increase in lysozyme mRNA expression, indicating a synergistic cooperation of PKA and Ca2+. This synergistic effect of PKA and Ca2+ was observed on the expression of a stably integrated CAT construct, controlled by the lysozyme promoter and the -6.1-kb enhancer containing binding sites for C/EBP and NF-kappaB/Rel. Therefore, we discussed the role of C/EBPbeta(NF-M), CREB, and NF-kappaB/Rel as possible targets for phosphorylation mediated by PKA, PKC, and Ca2+.

  18. Combination of acoustic levitation with small angle scattering techniques and synchrotron radiation circular dichroism. Application to the study of protein solutions. (United States)

    Cristiglio, Viviana; Grillo, Isabelle; Fomina, Margarita; Wien, Frank; Shalaev, Evgenyi; Novikov, Alexey; Brassamin, Séverine; Réfrégiers, Matthieu; Pérez, Javier; Hennet, Louis


    The acoustic levitation technique is a useful sample handling method for small solid and liquids samples, suspended in air by means of an ultrasonic field. This method was previously used at synchrotron sources for studying pharmaceutical liquids and protein solutions using x-ray diffraction and small angle x-ray scattering (SAXS). In this work we combined for the first time this containerless method with small angle neutron scattering (SANS) and synchrotron radiation circular dichroism (SRCD) to study the structural behavior of proteins in solutions during the water evaporation. SANS results are also compared with SAXS experiments. The aggregation behavior of 45μl droplets of lysozyme protein diluted in water was followed during the continuous increase of the sample concentration by evaporating the solvent. The evaporation kinetics was followed at different drying stage by SANS and SAXS with a good data quality. In a prospective work using SRCD, we also studied the evolution of the secondary structure of the myoglobin protein in water solution in the same evaporation conditions. Acoustic levitation was applied for the first time with SANS and the high performances of the used neutron instruments made it possible to monitor fast container-less reactions in situ. A preliminary work using SRCD shows the potentiality of its combination with acoustic levitation for studying the evolution of the protein structure with time. This multi-techniques approach could give novel insights into crystallization and self-assembly phenomena of biological compound with promising potential applications in pharmaceutical, food and cosmetics industry. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Lysozyme stability and amyloid fibrillization dependence on Hofmeister anions in acidic pH. (United States)

    Poniková, Slavomíra; Antošová, Andrea; Demjén, Erna; Sedláková, Dagmar; Marek, Jozef; Varhač, Rastislav; Gažová, Zuzana; Sedlák, Erik


    We have explored an effect of Hofmeister anions, Na2SO4, NaCl, NaBr, NaNO3, NaSCN and NaClO4, on stability and amyloid fibrillization of hen egg white lysozyme at pH 2.7. The stability of the protein was analyzed by differential scanning calorimetry. The Hofmeister effect of the anions was assessed by the parameter dT trs/d[anion] (T trs, transition temperature). We show that dT trs/d[anion] correlates with anion surface tension effects and anion partition coefficients indicating direct interactions between anions and lysozyme. The kinetic of amyloid fibrillization of lysozyme was followed by Thioflavin T (ThT) fluorescence. Negative correlation between dT trs/d[anion] and the nucleation rate of fibrillization in the presence of monovalent anions indicates specific effect of anions on fibrillization rate of lysozyme. The efficiency of monovalent anions to accelerate fibrillization correlates with inverse Hofmeister series. The far-UV circular dichroism spectroscopy and atomic force microscopy findings show that conformational properties of fibrils depend on fibrillization rate. In the presence of sodium chloride, lysozyme forms typical fibrils with elongated structure and with the secondary structure of the β-sheet. On the other hand, in the presence of both chaotropic perchlorate and kosmotropic sulfate anions, the fibrils form clusters with secondary structure of β-turn. Moreover, the acceleration of fibril formation is accompanied by decreased amount of the formed fibrils as indicated by ThT fluorescence. Taken together, our study shows Hofmeister effect of monovalent anions on: (1) lysozyme stability; (2) ability to accelerate nucleation phase of lysozyme fibrillization; (3) amount, and (4) conformational properties of the formed fibrils.

  20. Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue

    Directory of Open Access Journals (Sweden)

    Ming Feng Jiang


    Full Text Available Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type milk lysozyme gene (YML, was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75 which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity.

  1. XtalView, protein structure solution and protein graphics, a short history. (United States)

    McRee, Duncan E; Israel, Mark


    From a user's point-of-view we are in the Golden Age of protein crystallographic software. In the past few decades, solving protein structures has gone from a task requiring man-months of effort to a process requiring minutes on an ordinary laptop with no human intervention required. The birth of XtalView coincided with the mainstream use of synchrotron radiation, seleno-Met phasing and it continues to be used in this age of robotic crystallization, Fed-Ex data collection and fully automated structure solution "pipelines". This article is a retrospective history of protein crystallographic computing and a discussion of the current state of the art.

  2. Laser ablation dynamics and production of thin films of lysozyme

    DEFF Research Database (Denmark)

    Canulescu, Stela; Schou, Jørgen; Amoruso, S.;

    produced thin films of average thickness up to 300 nm, which not only contained a significant amount of intact molecules, but also maintained the bioactivity. These films were produced by a nanosecond laser in the UV regime at 355 nm with 2 J/cm2. The surprising fact that these molecules can be transferred....... This is the first time the ablation by fs-lasers of a protein has been recorded quantitatively. Films of lysozyme produced by fs-laser irradiation were analyzed by MALDI and a significant number of intact molecules in the films with fs-laser deposition was found as well....

  3. Dataset concerning GroEL chaperonin interaction with proteins

    Directory of Open Access Journals (Sweden)

    V.V. Marchenkov


    Full Text Available GroEL chaperonin is well-known to interact with a wide variety of polypeptide chains. Here we show the data related to our previous work ( [1], and concerning the interaction of GroEL with native (lysozyme, α-lactalbumin and denatured (lysozyme, α-lactalbumin and pepsin proteins in solution. The use of affinity chromatography on the base of denatured pepsin for GroEL purification from fluorescent impurities is represented as well.

  4. Study of lysozyme resistance in Rhodococcus equi. (United States)

    Hébert, Laurent; Bidaud, Pauline; Goux, Didier; Benachour, Abdellah; Laugier, Claire; Petry, Sandrine


    Lysozyme is an important and widespread component of the innate immune response that constitutes the first line of defense against bacterial pathogens. The bactericidal effect of this enzyme relies on its capacity to hydrolyze the bacterial cell wall and also on a nonenzymatic mechanism involving its cationic antimicrobial peptide (CAMP) properties, which leads to membrane permeabilization. In this paper, we report our findings on the lysozyme resistance ability of Rhodococcus equi, a pulmonary pathogen of young foals and, more recently, of immunocompromised patients, whose pathogenic capacity is conferred by a large virulence plasmid. Our results show that (i) R. equi can be considered to be moderately resistant to lysozyme, (ii) the activity of lysozyme largely depends on its muramidase action rather than on its CAMP activity, and (iii) the virulence plasmid confers part of its lysozyme resistance capacity to R. equi. This study is the first one to demonstrate the influence of the virulence plasmid on the stress resistance capacity of R. equi and improves our understanding of the mechanisms enabling R. equi to resist the host defenses.

  5. Solvent Electrostatic Response: From Simple Solutes to Proteins (United States)

    Dinpajooh, Mohammadhasan

    How water behaves at interfaces is relevant to many scientific and technological applications; however, many subtle phenomena are unknown in aqueous solutions. In this work, interfacial structural transition in hydration shells of a polarizable solute at critical polarizabilities is discovered. The transition is manifested in maximum water response, the reorientation of the water dipoles at the interface, and an increase in the density of dangling OH bonds. This work also addresses the role of polarizability of the active site of proteins in biological catalytic reactions. For proteins, the hydration shell becomes very heterogeneous and involves a relatively large number of water molecules. The molecular dynamics simulations show that the polarizability, along with the atomic charge distribution, needs to be a part of the picture describing how enzymes work. Non Gaussian dynamics in time-resolved linear and nonlinear (correlation) 2D spectra are also analyzed. Additionally, a theoretical formalism is presented to show that when preferential orientations of water dipoles exist at the interface, electrophoretic charges can be produced without free charge carriers, i.e., neutral solutes can move in a constant electric field due to the divergence of polarization at the interface. Furthermore, the concept of interface susceptibility is introduced. It involves the fluctuations of the surface charge density caused by thermal motion and its correlation over the characteristic correlation length with the fluctuations of the solvent charge density. Solvation free energy and interface dielectric constant are formulated accordingly. Unlike previous approaches, the solvation free energy scales quite well in a broad range of ion sizes, namely in the range of 2-14 A. Interface dielectric constant is defined such that the boundary conditions in the Laplace equation describing a micro- or mesoscopic interface are satisfied. The effective dielectric constant of interfacial water is

  6. Non-ideal behavior of binary aqueous mixtures of some urea derivatives and their capacity to induce lysozyme gelation. (United States)

    de Souza, Ícaro F T; Arêas, Elizabeth P G


    The urea derivatives, namely, ethylurea (EU), 1,3 dimethylurea (1,3-DMU) and 1,1 diethylurea (1,1-DEU), in the limiting regions of their solubilities in water, and tetramethylurea (TMU) at w≥0.65 were investigated in relation to their capacity of inducing hen egg white lysozyme (HEWL) physical (non-covalent) gelation. Protein transparent gels were generated out of TMU/H2O and 1,1-DEU/H2O, respectively, whereas an intensively turbid gel resulted from sol-gel transition taking place in EU/H2O. Oscillatory rheology revealed distinctions in the gels' structural and dynamic characteristics. Hydration patterns of the derivatives in solution, sizes of their non-polar domains and supramolecular symmetry features played a central role in their capacity of gel formation and in the gels' rheological behavior and morphology. Effects on gel characteristics of distinctively positioned ions in the Hofmeister series showed that SCN(-) disrupted water H-bonding interconnectivity in TMU lysozyme gel, strengthening gel structure, yet maintaining gel transparency. Citrate enhanced system elasticity albeit causing intense turbidity and leading to phase separation. Larger values of the storage modulus, G', were verified for gels generated from binary mixtures containing urea derivatives with higher dipole moments. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Interaction of fullerenol with lysozyme investigated by experimental and computational approaches

    Energy Technology Data Exchange (ETDEWEB)

    Yang Shengtao; Wang Haifang; Guo Lin; Gao Yang; Liu Yuanfang [Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Cao Aoneng [Institute of Nanochemistry and Nanobiology, Shanghai University, Shanghai 200444 (China)], E-mail:, E-mail:


    The potential biomedical applications of fullerenol C{sub 60}(OH){sub x} (x{approx}24) have been extensively studied. However, the structural information of the interaction of fullerenol with the bio-system at the molecular level, which is essential for understanding its bioactivity and toxicity, is still missing. In this study, lysozyme was selected as a model protein to investigate the interaction between fullerenol and biomolecules. A strong induced circular dichroism (CD) signal of achiral fullerenol was observed after binding with lysozyme. Activity assay shows that lysozyme activity is inhibited significantly by fullerenol. No heat capacity difference between the folded and unfolded states of lysozyme was measured by differential scanning calorimetry (DSC) in the presence of fullerenol, indicating that fullerenol prefers to bind with the hydrophobic residues. Both experimental and Autodock computational results suggest that the binding site on lysozyme for fullerenol is close to Trp 62, and a {pi}-{pi} stacking interaction might play an important role in binding.

  8. Binding and Inhibitory Effect of the Dyes Amaranth and Tartrazine on Amyloid Fibrillation in Lysozyme. (United States)

    Basu, Anirban; Suresh Kumar, Gopinatha


    Interaction of two food colorant dyes, amaranth and tartrazine, with lysozyme was studied employing multiple biophysical techniques. The dyes exhibited hypochromic changes in the presence of lysozyme. The intrinsic fluorescence of lysozyme was quenched by both dyes; amaranth was a more efficient quencher than tartrazine. The equilibrium constant of amaranth was higher than that of tartarzine. From FRET analysis, the binding distances for amaranth and tartrazine were calculated to be 4.51 and 3.93 nm, respectively. The binding was found to be dominated by non-polyelectrolytic forces. Both dyes induced alterations in the microenvironment surrounding the tryptophan and tyrosine residues of the protein, with the alterations being comparatively higher for the tryptophans than the tyrosines. The interaction caused significant loss in the helicity of lysozyme, the change being higher with amaranth. The binding of both dyes was exothermic. The binding of amaranth was enthalpy driven, while that of tartrazine was predominantly entropy driven. Amaranth delayed lysozyme fibrillation at 25 μM, while tartrazine had no effect even at 100 μM. Nevertheless, both dyes had a significant inhibitory effect on fibrillogenesis. The present study explores the potential antiamyloidogenic property of these azo dyes used as food colorants.

  9. Neutron crystallographic studies of T4 lysozyme at cryogenic temperature. (United States)

    Li, Le; Shukla, Shantanu; Meilleur, Flora; Standaert, Robert F; Pierce, Josh; Myles, Dean A A; Cuneo, Matthew J


    Bacteriophage T4 lysozyme (T4L) has been used as a paradigm for seminal biophysical studies on protein structure, dynamics, and stability. Approximately 700 mutants of this protein and their respective complexes have been characterized by X-ray crystallography; however, despite the high resolution diffraction limits attained in several studies, no hydrogen atoms were reported being visualized in the electron density maps. To address this, a 2.2 Å-resolution neutron data set was collected at 80 K from a crystal of perdeuterated T4L pseudo-wild type. We describe a near complete atomic structure of T4L, which includes the positions of 1737 hydrogen atoms determined by neutron crystallography. The cryogenic neutron model reveals explicit detail of the hydrogen bonding interactions in the protein, in addition to the protonation states of several important residues. © 2017 The Protein Society.

  10. Influence of some formulation and process parameters on the stability of lysozyme incorporated in corn flour- or corn starch-based extruded materials prepared by melt blending processing. (United States)

    Jbilou, Fouzia; Galland, Sophie; Telliez, Camille; Akkari, Zied; Roux, Roselyne; Oulahal, Nadia; Dole, Patrice; Joly, Catherine; Degraeve, Pascal


    In order to obtain an antimicrobial biodegradable material, corn flour was extruded with 1% of lysozyme. Since the limited stability of natural preservatives such as lysozyme is a common bottleneck to the elaboration of active biomaterials by melt blending processes, the influence of formulation and of extrusion processing temperature on its residual enzymatic activity was investigated. To assess the contribution of process parameters such as temperature, shear stress and of related formulation parameters such as glycerol and moisture contents, the stability of lysozyme following its extrusion or its thermoforming with plasticized corn starch or thermal treatments in aqueous glycerol solutions was also studied. Increasing glycerol content from 25% to 30% significantly limited inactivation of lysozyme during extrusion, while increasing initial moisture content of the mixture from 14.5% to 28.5% had the opposite effect. These observations open the possibility to prepare active materials retaining more than 60±7% of initial lysozyme activity.

  11. Grafted block complex coacervate core micelles and their effect on protein adsorption on silica and polystyrene

    NARCIS (Netherlands)

    Brzozowska, Agata M.; de Keizer, Arie; Norde, Willem; Detrembleur, Christophe; Stuart, Martien Cohen


    We have studied the formation and the stability of grafted block complex coacervate core micelles (C3Ms) in solution and the influence of grafted block C3M coatings on the adsorption of the proteins beta-lactoglobulin, bovine serum albumin, and lysozyme. The C3Ms consist of a grafted block copolymer

  12. Hydrophobic nano-carrier for lysozyme adsorption

    Indian Academy of Sciences (India)



    In this work, poly(HEMA–APH) nanoparticles were synthesized by surfactant-free emulsion polymerization technique.Magnetic behaviour was introduced by simple addition of Fe$_3$O$_4$ into the polymerization medium.Characterization of the nanoparticle was carried out by FTIR, ESR, SEM, AFM and EDX analyses. These synthesized magnetic nanoparticles were used for adsorption of lysozyme. For this purpose, adsorption conditions wereoptimized and maximum lysozyme binding capacity was found to be 278.8 mg g$^{−1}$ polymer in pH 7.0 phosphate buffer at 25$^{\\circ}$C. Desorption and reusability properties of the nanoparticles were investigated and lysozyme adsorption efficiency did not change significantly at the end of the 10 successive reuses.

  13. Heat-denatured lysozyme aggregation and gelation as revealed by combined dielectric relaxation spectroscopy and light scattering measurements. (United States)

    Giugliarelli, A; Sassi, P; Paolantoni, M; Onori, G; Cametti, C


    The dielectric behavior of native and heat-denatured lysozyme in ethanol-water solutions was examined in the frequency range from 1 MHz to 2 GHz, using frequency-domain dielectric relaxation spectroscopy. Because of the conformational changes on unfolding, dielectric methods provide information on the denaturation process of the protein and, at protein concentration high enough, on the subsequent aggregation and gelation. Moreover, the time evolution of the protein aggregation and gelation was monitored measuring, by means of dynamic light scattering methods, the diffusion coefficient of micro-sized polystyrene particles, deliberately added to the protein solution, which act as a probe of the viscosity of the microenvironment close to the particle surface. All together, our measurements indicate that heat-induced denaturation favors, at high concentrations, a protein aggregation process which evolves up to the full gelation of the system. These findings have a direct support from IR measurements of the absorbance of the amide I band that, because of the unfolding, indicate that proteins entangle each other, producing a network structure which evolves, in long time limit, in the gel.

  14. Superheating of ice crystals in antifreeze protein solutions (United States)

    Celik, Yeliz; Graham, Laurie A.; Mok, Yee-Foong; Bar, Maya; Davies, Peter L.; Braslavsky, Ido


    It has been argued that for antifreeze proteins (AFPs) to stop ice crystal growth, they must irreversibly bind to the ice surface. Surface-adsorbed AFPs should also prevent ice from melting, but to date this has been demonstrated only in a qualitative manner. Here we present the first quantitative measurements of superheating of ice in AFP solutions. Superheated ice crystals were stable for hours above their equilibrium melting point, and the maximum superheating obtained was 0.44 °C. When melting commenced in this superheated regime, rapid melting of the crystals from a point on the surface was observed. This increase in melting temperature was more appreciable for hyperactive AFPs compared to the AFPs with moderate antifreeze activity. For each of the AFP solutions that exhibited superheating, the enhancement of the melting temperature was far smaller than the depression of the freezing temperature. The present findings clearly show that AFPs adsorb to ice surfaces as part of their mechanism of action, and this absorption leads to protection of ice against melting as well as freezing. PMID:20215465

  15. Superheating of ice crystals in antifreeze protein solutions. (United States)

    Celik, Yeliz; Graham, Laurie A; Mok, Yee-Foong; Bar, Maya; Davies, Peter L; Braslavsky, Ido


    It has been argued that for antifreeze proteins (AFPs) to stop ice crystal growth, they must irreversibly bind to the ice surface. Surface-adsorbed AFPs should also prevent ice from melting, but to date this has been demonstrated only in a qualitative manner. Here we present the first quantitative measurements of superheating of ice in AFP solutions. Superheated ice crystals were stable for hours above their equilibrium melting point, and the maximum superheating obtained was 0.44 degrees C. When melting commenced in this superheated regime, rapid melting of the crystals from a point on the surface was observed. This increase in melting temperature was more appreciable for hyperactive AFPs compared to the AFPs with moderate antifreeze activity. For each of the AFP solutions that exhibited superheating, the enhancement of the melting temperature was far smaller than the depression of the freezing temperature. The present findings clearly show that AFPs adsorb to ice surfaces as part of their mechanism of action, and this absorption leads to protection of ice against melting as well as freezing.

  16. Uptake and release kinetics of lysozyme in and from an oxidized starch polymer microgel

    NARCIS (Netherlands)

    Li, Y.; Zhang, Z.; Leeuwen, van H.P.; Cohen Stuart, M.A.; Norde, W.; Kleijn, J.M.


    The kinetics of uptake and release of fluorescently labeled lysozyme by/from spherical oxidized starch polymer microgel particles (diameter 10–20 µm) was investigated using confocal laser scanning microscopy. Both the protein and the microgel have a pH dependent charge; in the pH range 3–9, the prot

  17. Uptake and release kinetics of lysozyme in and from an oxidized starch polymer microgel

    NARCIS (Netherlands)

    Li, Yuan; Zhang, Zeshi; van Leeuwen, Herman P.; Stuart, Martien A. Cohen; Norde, Willem; Kleijn, J. Mieke


    The kinetics of uptake and release of fluorescently labeled lysozyme by/from spherical oxidized starch polymer microgel particles (diameter 10-20 mu m) was investigated using confocal laser scanning microscopy. Both the protein and the microgel have a pH dependent charge; in the pH range 3-9, the pr

  18. Pluronic-lysozyme conjugates as anti-adhesive and antibacterial bifunctional polymers for surface coating

    NARCIS (Netherlands)

    Muszanska, A.K.; Busscher, H.J.; Herrmann, A.; Mei, van der H.C.; Norde, W.


    This paper describes the preparation and characterization of polymer protein conjugates composed of a synthetic triblock copolymer with a central polypropylene oxide (PPO) block and two terminal polyethylene oxide (PEO) segments, Pluronic F-127, and the antibacterial enzyme lysozyme attached to the

  19. Pluronic-lysozyme conjugates as anti-adhesive and antibacterial bifunctional polymers for surface coating

    NARCIS (Netherlands)

    Muszanska, Agnieszka K.; Busscher, Henk J.; Herrmann, Andreas; van der Mei, Henny C.; Norde, Willem

    This paper describes the preparation and characterization of polymer protein conjugates composed of a synthetic triblock copolymer with a central polypropylene oxide (PPO) block and two terminal polyethylene oxide (PEO) segments, Pluronic F-127, and the antibacterial enzyme lysozyme attached to the

  20. Pluronic-lysozyme conjugates as anti-adhesive and antibacterial bifunctional polymers for surface coating

    NARCIS (Netherlands)

    Muszanska, A.K.; Busscher, H.J.; Herrmann, A.; Mei, van der H.C.; Norde, W.


    This paper describes the preparation and characterization of polymer protein conjugates composed of a synthetic triblock copolymer with a central polypropylene oxide (PPO) block and two terminal polyethylene oxide (PEO) segments, Pluronic F-127, and the antibacterial enzyme lysozyme attached to the

  1. The Effect of Ethylene Glycol, Glycine Betaine, and Urea on Lysozyme Thermal Stability (United States)

    Schwinefus, Jeffrey J.; Leslie, Elizabeth J.; Nordstrom, Anna R.


    The four-week student project described in this article is an extension of protein thermal denaturation experiments to include effects of added cosolutes ethylene glycol, glycine betaine, and urea on the unfolding of lysozyme. The transition temperatures and van't Hoff enthalpies for unfolding are evaluated for six concentrations of each cosolute,…

  2. The Effect of Ethylene Glycol, Glycine Betaine, and Urea on Lysozyme Thermal Stability (United States)

    Schwinefus, Jeffrey J.; Leslie, Elizabeth J.; Nordstrom, Anna R.


    The four-week student project described in this article is an extension of protein thermal denaturation experiments to include effects of added cosolutes ethylene glycol, glycine betaine, and urea on the unfolding of lysozyme. The transition temperatures and van't Hoff enthalpies for unfolding are evaluated for six concentrations of each cosolute,…

  3. Renal-selective delivery and angiotensin-converting enzyme inhibition by subcutaneously administered captopril-lysozyme

    NARCIS (Netherlands)

    Prakash, Jai; van Loenen - Weemaes, Anne-miek; Haas, M; Proost, Hans; Meijer, D.K F; Moolenaar, Frits; Poelstra, Klaas; Kok, R.J

    In previous studies, we have demonstrated that the low molecular weight protein lysozyme can be used as a renal-selective drug carrier for delivery of the angiotensin-converting enzyme ( ACE) inhibitor captopril. Typically, such macromolecular drug-targeting preparations are administered

  4. Study on flow characteristics of solid/liquid system in lysozyme crystal growth

    Institute of Scientific and Technical Information of China (English)

    CUI HaiLiang; YU Yong; CHEN WanChun; KANG Qi


    During the process of lysozyme protein crystallization with batch method, the macroscopic flow field of solid/liquid system was observed by particle image velocimetry (PIV). Furthermore, a normal growth rate of (110) face and local flow field around a single protein crystal were obtained by a long work distance microscope. The experimental results showed that the average velocity, the maximal velocity of macroscopic solid/liquid system and the velooity of local flow field around single protein crystal were fluctuant. The effective boundary layer thickness δeff, the concentration at the interface Gi and the characteristic velocity V were calculated using a convection-diffusion model. The results showed that the growth of lysozyme crystal in this experiment was dominated by interfacial kinetics rather than bulk transport, and the function of buoyancy-driven flow in bulk transport was small, however, the effect of bulk transport in crystal growth had a tendency to increase with the increase of lysozyme concentration. The calculated results also showed that the order of magnitude of shear force was about 10-21 N,which was much less than the bond force between the lysozyme molecules. Therefore the shear force induced by buoyancy-driven flows cannot remove the protein molecules from the interface of crystal.

  5. Lysozyme Thermal Denaturation and Self-Interaction: Four Integrated Thermodynamic Experiments for the Physical Chemistry Laboratory (United States)

    Schwinefus, Jeffrey J.; Schaefle, Nathaniel J.; Muth, Gregory W.; Miessler, Gary L.; Clark, Christopher A.


    As part of an effort to infuse our physical chemistry laboratory with biologically relevant, investigative experiments, we detail four integrated thermodynamic experiments that characterize the denaturation (or unfolding) and self-interaction of hen egg white lysozyme as a function of pH and ionic strength. Students first use Protein Explorer to…

  6. Uptake and release kinetics of lysozyme in and from an oxidized starch polymer microgel

    NARCIS (Netherlands)

    Li, Y.; Zhang, Z.; Leeuwen, van H.P.; Cohen Stuart, M.A.; Norde, W.; Kleijn, J.M.


    The kinetics of uptake and release of fluorescently labeled lysozyme by/from spherical oxidized starch polymer microgel particles (diameter 10–20 µm) was investigated using confocal laser scanning microscopy. Both the protein and the microgel have a pH dependent charge; in the pH range 3–9, the prot

  7. Uptake and release kinetics of lysozyme in and from an oxidized starch polymer microgel

    NARCIS (Netherlands)

    Li, Yuan; Zhang, Zeshi; van Leeuwen, Herman P.; Stuart, Martien A. Cohen; Norde, Willem; Kleijn, J. Mieke


    The kinetics of uptake and release of fluorescently labeled lysozyme by/from spherical oxidized starch polymer microgel particles (diameter 10-20 mu m) was investigated using confocal laser scanning microscopy. Both the protein and the microgel have a pH dependent charge; in the pH range 3-9, the pr

  8. Ultra-sensitive quantification of lysozyme based on element chelate labeling and capillary electrophoresis–inductively coupled plasma mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Yang, MingWei [Key Laboratory of Analysis and Detection for Food Safety of Ministry of Education, Fujian Provincial Key Lab of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350108, Fujian (China); Department of Chemistry, Florida International University, Miami, FL 33199 (United States); Wu, WeiHua; Ruan, YaJuan; Huang, LiMei [Key Laboratory of Analysis and Detection for Food Safety of Ministry of Education, Fujian Provincial Key Lab of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350108, Fujian (China); Wu, Zujian [Department of Plant Protection, Fujian Agriculture and Forest University, Fuzhou 350002, Fujian (China); Cai, Yong [Department of Chemistry, Florida International University, Miami, FL 33199 (United States); Fu, FengFu, E-mail: [Key Laboratory of Analysis and Detection for Food Safety of Ministry of Education, Fujian Provincial Key Lab of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350108, Fujian (China); Department of Plant Protection, Fujian Agriculture and Forest University, Fuzhou 350002, Fujian (China)


    Graphical abstract: An ultra-sensitive method for the determination of lysozyme was developed based on the Gd{sup 3+} chelate labeling and CE–ICP–MS. The proposed method has an extremely low detection limit of 3.89 attomole and has been successfully used to detect lysozyme in saliva sample, showing excellent reliability. The success of the present method provides a new possibility for biological assays and clinical diagnoses. -- Highlights: •An ultra-sensitive method for detecting lysozyme based on CE–ICP–MS was described. •The proposed method has an extremely low detection limit of 3.89 attomole. •It can be used to detect trace lysozyme in saliva sample with a satisfied recovery. •The method provides a new potential for sensitive detection of low-abundant proteins. -- Abstract: In this study, an ultra-sensitive method for the quantification of lysozyme based on the Gd{sup 3+} diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid labeling and capillary electrophoresis–inductively coupled plasma mass spectrometry (CE–ICP–MS) was described. The Gd{sup 3+}-tagged lysozyme was effectively separated by capillary electrophoresis (CE) and sensitively determined by inductively coupled plasma mass spectrometry (ICP–MS). Based on the gadolinium-tagging and CE–ICP–MS, the lysozyme was determined within 12 min with an extremely low detection limit of 3.89 attomole (3.89 × 10{sup −11} mol L{sup −1} for 100 nL of sample injection) and a RSD < 6% (n = 5). The proposed method has been successfully used to detect lysozyme in saliva samples with a recovery of 91–106%, suggesting that our method is sensitive and reliable. The success of the present method provides a new potential for the biological assays and sensitive detection of low-abundant proteins.

  9. Buffer Effects in the Solubility, Nucleation and Growth of Chicken Egg White Lysozyme (United States)

    Gibson, Ursula J.


    The growth of protein crystals is important for determination of their three-dimensional structure, which relates to their biochemical functions and to the practical goal of designing pharmaceuticals to modify that function. While many proteins have been successfully crystallized by a variety of methods, there is still limited understanding of the process of nucleation and growth of even the simplest proteins. Chicken egg-white lysozyme (CEWL) is readily crystallized under a variety of conditions, and studies underway at MSFC are designed to elucidate the mechanisms by which the crystals nucleate and grow. We have investigated the effect of buffer choice on the solubility, nucleation and growth of CEWL. CEWL was purified by dialysis against a .05M phosphate buffer and chromatographic separation from contaminants in a sepharose column. Solubility studies were made as a function of buffer concentration for phosphate and formate buffers, and the nucleation and growth of crystals at 10 C was studied as a function of pH for oxalate, succinate, formate, butyrate, carbonate, phosphate and acetate buffer solutions. The solubility data support the conclusion that there is a solubility minimum as a function of buffer concentration for amphiphilic molecules, while no minimum is observed for a phosphate buffer. Nucleation is suppressed at pH greater than pKa for all buffers except phosphate. The aspect ratio of the (110) faces is shown to be a function of crystal size, rather than pH.

  10. Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation

    KAUST Repository

    Shah, Dhawal


    We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ XP, contrary to conventional thinking that positive Γ XP\\'s indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine\\'s preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine\\'s interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ XP, which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation. © 2011 American Institute of Chemical Engineers (AIChE).

  11. Recombinant goose-type lysozyme in channel catfish: Lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection (United States)

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme g (CC-Lys-g) produced in E. coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme g plasmid DNA could be used as an immunostimulant to protect chann...

  12. Hydration dynamics of protein molecules in aqueous solution: Unity among diversity

    Indian Academy of Sciences (India)

    Biman Jana; Subrata Pal; Biman Bagchi


    Dielectric dispersion and NMRD experiments have revealed that a significant fraction of water molecules in the hydration shell of various proteins do not exhibit any slowing down of dynamics. This is usually attributed to the presence of the hydrophobic residues (HBR) on the surface, although HBRs alone cannot account for the large amplitude of the fast component. Solvation dynamics experiments and also computer simulation studies, on the other hand, repeatedly observed the presence of a non-negligible slow component. Here we show, by considering three well-known proteins (lysozyme, myoglobin and adelynate kinase), that the fast component arises partly from the response of those water molecules that are hydrogen bonded with the backbone oxygen (BBO) atoms. These are structurally and energetically less stable than those with the side chain oxygen (SCO) atoms. In addition, the electrostatic interaction energy distribution (EIED) of individual water molecules (hydrogen bonded to SCO) with side chain oxygen atoms shows a surprising two peak character with the lower energy peak almost coincident with the energy distribution of water hydrogen bonded to backbone oxygen atoms (BBO). This two peak contribution appears to be quite general as we find it for lysozyme, myoglobin and adenylate kinase (ADK). The sharp peak of EIED at small energy (at less than 2 kBT) for the BBO atoms, together with the first peak of EIED of SCO and the HBRs on the protein surface, explain why a large fraction (∼ 80%) of water in the protein hydration layer remains almost as mobile as bulk water. Significant slowness arises only from the hydrogen bonds that populate the second peak of EIED at larger energy (at about 4 kBT). Thus, if we consider hydrogen bond interaction alone, only 15-20% of water molecules in the protein hydration layer can exhibit slow dynamics, resulting in an average relaxation time of about 5-10 ps. The latter estimate assumes a time constant of 20-100 ps for the slow

  13. Plasma graft of poly(ethylene glycol) methyl ether methacrylate (PEGMA) on RGP lens surface for reducing protein adsorption (United States)

    Shiheng, Yin; Li, Ren; Yingjun, Wang


    Poly(ethylene glycol) methyl ether methacrylate (PEGMA) was grafted on fluorosilicone acrylate rigid gas permissible contact lens surface by means of argon plasma induced polymerization to improve surface hydrophilicity and reduce protein adsorption. The surface properties were characterized by contact angle measurement, x-ray photoelectron spectroscopy (XPS) and atomic force microscopy respectively. The surface protein adsorption was evaluated by lysozyme solution immersion and XPS analysis. The results indicated that a thin layer of PEGMA was successfully grafted. The surface hydrophilicity was bettered and surface free energy increased. The lysozyme adsorption on the lens surface was reduced greatly. The study was supported by National Natural Science Foundation of China (No. 51273072).

  14. Relation between the solubility of proteins in aqueous solutions and the second virial coefficient of the solution

    NARCIS (Netherlands)

    Haas, C; Drenth, J; Wilson, WW


    Tn recent publications it was pointed out that there is a correlation between the observed values of the solubility of proteins in aqueous solutions and the second virial coefficient of the solution. In this paper we give a theoretical explanation of this relation. The derived theoretical expression

  15. Relation between the solubility of proteins in aqueous solutions and the second virial coefficient of the solution

    NARCIS (Netherlands)

    Haas, C; Drenth, J; Wilson, WW


    Tn recent publications it was pointed out that there is a correlation between the observed values of the solubility of proteins in aqueous solutions and the second virial coefficient of the solution. In this paper we give a theoretical explanation of this relation. The derived theoretical expression

  16. Influenceof lysozyme-like protein on virulence and capsular polysaccharide synthesis of Streptococcus pneumoniae%一种溶菌酶样蛋白对肺炎链球菌毒力及荚膜多糖合成的影响

    Institute of Scientific and Technical Information of China (English)

    黄健; 姜学美; 黄美容; 杨晓亮; 刘明伟; 王虹; 孟江萍


    [Objective]To study the biological character and pathogenic effect of a lysozyme-like protein in Streptococcus pneumoniae.[Methods]The gene knockout mutant was constructed by the long flanking homology polymerase chain reaction.The mutant containing rescue plasmid to complement the lysozyme-like gene was also constructed.The differences in biology and pathogenicity in D39 wild strain, gene knockout mutant and gene knockout mutant containing rescue plasmid were observed to identify the functions of the lysozyme-like gene.[Results]Compared with the wild strain, the gene knockout mutant had the characteristics of slower growth, declining virulence, and obviously reduced capsule polysaccharide synthesis.Complement experiment showed when the rescue plasmid was transformed into the mutant strain, the mRNA level of hypothetical lysozyme-like gene in the gene knockout mutant containing rescue plasmid was higher than that of the wild strain.Although the levels of virulence and capsule polysaccharide synthesis could be partly complemented compared with those of the gene knockout mutant, but not reached the levels in the wild strain.[Conclusions]The lysozyme-like protein, a new Streptococcus pneumoniae virulence factor, may regulate the proliferation and the capsular polysaccharides synthesis of Streptococcus pneumoniae to affect expression of virulence.%[目的]为了研究肺炎链球菌(Streptococcus pneumoniae,的一种假想的溶菌酶样蛋白在细菌生物学性状及其致病中的作用.[方法]利用长臂同源PCR对该基因进行敲出,并同时构建带有拯救质粒的缺失菌株,观察D39野生菌、缺失菌与带有拯救质粒的缺失菌株在相关生物学性状及其致病力改变,从而鉴定这种假想溶菌酶样蛋白的功能.[结果]缺失菌与野生菌相比,细菌生长减缓,毒力下降,荚膜多糖合成明显减少.而将拯救质粒转入缺失菌株后,该溶菌酶样蛋白的mRNA表达水平较野生菌高,其毒力及荚膜

  17. Physicochemical characterization and sorption behavior of Mg-Ca-Al (NO3) hydrotalcite-like compounds toward removal of fluoride from protein solutions. (United States)

    Lv, Tengfei; Ma, Wei; Xin, Gang; Wang, Ren; Xu, Jun; Liu, Dongmei; Liu, Fujun; Pan, Decong


    The present study explores the potential of Mg-Ca-Al (NO(3)) hydrotalcite-like compounds (MgCaAlNO(3)-HTlcs) for the removal of fluoride from protein solutions. In this study, the Mg(3-x)Ca(x)AlNO(3)-HTlcs (x=0-3, x is the mol.% of Ca) were synthesized and characterized by SEM, XRD, FTIR, BET, ICP-AES and pHzpc analysis. The sorption experiments were conducted in protein systems of bovine serum albumin (BSA) and lysozyme (LSZ). The batch experiment results showed that the NO(3)-HTlc with Mg/Ca/Al molar ratio of 2.5/0.5/1 had remarkable fluoride sorption ability with maximum sorption capacities of 82.35 mg/g and 72.69 mg/g at pH 5.0 and 40°C in BSA and LSZ system, respectively. Moreover, the loss of BSA of 0.71% was low and there was no loss of LSZ. It was evident that the Mg(2.5)Ca(0.5)AlNO(3)-HTlc could selectively adsorb fluoride from protein solutions. The equilibrium sorption data fitted well to the Langmuir model and the kinetic data conformed to the pseudo-second-order model. Thermodynamic parameters (ΔG°, ΔH° and ΔS°) were evaluated and revealed that the sorption process was spontaneous and endothermic in nature. Furthermore, the results from Antarctic krill processing wastewater study confirmed the feasibility and practicality of the Mg(2.5)Ca(0.5)AlNO(3)-HTlc for fluoride removal in fluoride bearing protein system.

  18. Lysozyme and succinylated lysozyme as adsorbates and emulsifiers

    NARCIS (Netherlands)

    Veen, van der M.


    Many food products are emulsions, i.e., a system of two immiscible liquids of which the one is finely dispersed in the other. In many emulsions, a monolayer of protein molecules in the liquid/liquid interface prevents coalescence of the droplets. Therefore, in the formation of a protein-stabilized e

  19. Lysozyme and succinylated lysozyme as adsorbates and emulsifiers

    NARCIS (Netherlands)

    Veen, van der M.


    Many food products are emulsions, i.e., a system of two immiscible liquids of which the one is finely dispersed in the other. In many emulsions, a monolayer of protein molecules in the liquid/liquid interface prevents coalescence of the droplets. Therefore, in the formation of a protein-stabilized

  20. Phase equilibria for salt-induced lysozyme precipitation: Effect of salt concentration and pH


    Popova, E.; WATANABE, E. O.; PESSOA FILHO, P. A.; Maurer, G.


    The salt-induced precipitation of lysozyme from aqueous solutions was studied at 25 degrees C and various pH values by cloud-point investigations, precipitation experiments (analysing the compositions of the coexisting phases) and microscopic investigations of the precipitates. Sodium sulphate as well as ammonium sulphate were used to induce the precipitation. The experimental results are discussed and used to develop a scheme of the phase equilibrium in water-rich aqueous solutions of lysozy...

  1. Concentration dependent switch in the kinetic pathway of lysozyme fibrillation: Spectroscopic and microscopic analysis (United States)

    Kiran Kumar, E.; Prasad, Deepak Kumar; Prakash Prabhu, N.


    Formation of amyloid fibrils is found to be a general tendency of many proteins. Investigating the kinetic mechanisms and structural features of the intermediates and the final fibrillar state is essential to understand their role in amyloid diseases. Lysozyme, a notable model protein for amyloidogenic studies, readily formed fibrils in vitro at neutral pH in the presence of urea. It, however, showed two different kinetic pathways under varying urea concentrations when probed with thioflavin T (ThT) fluorescence. In 2 M urea, lysozyme followed a nucleation-dependent fibril formation pathway which was not altered by varying the protein concentration from 2 mg/ml to 8 mg/ml. In 4 M urea, the protein exhibited concentration dependent change in the mechanism. At lower protein concentrations, lysozyme formed fibrils without any detectable nuclei (nucleation-independent polymerization pathway). When the concentration of the protein was increased above 3 mg/ml, the protein followed nucleation-dependent polymerization pathway as observed in the case of 2 M urea condition. This was further verified using microscopic images of the fibrils. The kinetic parameters such as lag time, elongation rate, and fibrillation half-time, which were derived from ThT fluorescence changes, showed linear dependency against the initial protein concentration suggested that under the nucleation-dependent pathway conditions, the protein followed primary-nucleation mechanism without any significant secondary nucleation events. The results also suggested that the differences in the initial protein conformation might alter the mechanism of fibrillation; however, at the higher protein concentrations lysozyme shifted to nucleation-dependent pathway.

  2. Structural characteristics and plant-beneficial effects of bacteria colonizing the shoots of field grown conventional and genetically modified T4-lysozyme producing potatoes

    NARCIS (Netherlands)

    Rasche, F.; Marco-Noales, E.; Velvis, H.; Overbeek, van L.S.; Lopez, M.M.; Elsas, van J.D.; Sessitsch, A.


    Genetically modified potatoes expressing antibacterial protein T4 lysozyme may offer effective control strategies for bacterial pathogens causing severe potato diseases. Apart from this beneficial effect, it is very important to investigate such engineered potatoes carefully for potential adverse ef

  3. Structural characteristics and plant-beneficial effects of bacteria colonizing the shoots of field grown conventional and genetically modified T4-lysozyme producing potatoes

    NARCIS (Netherlands)

    Rasche, Frank; Marco-Noales, Ester; Velvis, Henk; van Overbeek, Leo S.; Lopez, Maria M.; van Elsas, Jan D.; Sessitsch, Angela


    Genetically modified potatoes expressing antibacterial protein T4 lysozyme may offer effective control strategies for bacterial pathogens causing severe potato diseases. Apart from this beneficial effect, it is very important to investigate such engineered potatoes carefully for potential adverse ef

  4. Pr(Ⅲ) and Nd(Ⅲ) Absorption Spectroscopic Probe to Investigate Interaction with Lysozyme (HEW)

    Institute of Scientific and Technical Information of China (English)


    Pr(Ⅲ) and Nd(Ⅲ) can be utilized as absorption spectroscopic probes to investigate the interaction of biomolecules like Lysozyme (HEW) with Ca(Ⅱ) in-vitro; the most abundant metal ion in the human body system. The spectroscopic techniques involving comparative absorption, absorption difference, and quantitative intensity analysis using 4f-4f transitions are utilized for changes in the inner sphere coordination pattern of Pr(Ⅲ) and Nd(Ⅲ) in solution as well as in solid state. The present study deals with an important biomolecule in human metabolism, that is, Lysozyme (HEW). The absorption spectral parameters such as the oscillator strength (P), the Judd-Ofelt (Tλ) intensity parameters, and the Slater-Condon inter electronic parameters are calculated using chi square methods. The obtained results are used to determine the probable geometry of the complex in the solution, the nature of the bond between Pr(Ⅲ)/Nd(Ⅲ) with lysozyme, and the inner sphere coordination environment of f-f transitions. The results obtained from various experimental conditions are utilized to investigate the coordination changes in the Pr(Ⅲ)/Nd(Ⅲ) complexes caused by different coordinating sites of lysozyme, normalized bite, denticity, the solvent nature, the coordination number, the nature of bond and other parameters to mimic the interaction of the Ca(Ⅱ) ion with such biomolecule.

  5. Recent advances for the production and recovery methods of lysozyme. (United States)

    Ercan, Duygu; Demirci, Ali


    Lysozyme is an antimicrobial peptide with a high enzymatic activity and positive charges. Therefore, it has applications in food and pharmaceutical industries as an antimicrobial agent. Lysozyme is ubiquitous in both animal and plant kingdoms. Currently, egg-white lysozyme is the most commercially available form of lysozyme. The main concerns of egg-white lysozyme are high recovery cost, low activity and most importantly the immunological problems to some people. Therefore, human lysozyme production has gained importance in recent years. Scientists have developed transgenic plants, animals and microorganisms that can produce human lysozyme. Out of these, microbial production has advantages for commercial productions, because high production levels are achievable in a relatively short time. It has been reported that fermentation parameters, such as pH, temperature, aeration, are key factors to increase the effectiveness of the human lysozyme production. Moreover, purification of the lysozyme from the fermentation broth needs to be optimized for the economical production. In conclusion, this review paper covers the mechanism of lysozyme, its sources, production methods and recovery of lysozyme.

  6. Effects of cloud-point grafting, chain length, and density of PEG layers on competitive adsorption of ocular proteins

    DEFF Research Database (Denmark)

    Kingshott, P.; Thissen, H.; Griesser, H.J.


    The effects of pinning density, chain length, and 'cloud point' (CP) versus non-CP grafting conditions have been studied on the ability of polyethylene glycol (PEG) layers to minimize adsorption from a multicomponent (lysozyme, human serum albumin (HSA), IgG and lactoferrin) protein solution. Met...

  7. Effects of modified {beta}-cyclodextrin on thermal stability and conformation of lysozyme

    Energy Technology Data Exchange (ETDEWEB)

    Kamiyama, Tadashi, E-mail: [Department of Chemistry, School of Science and Engineering, Kinki University, Kowakae 3-4-1, Higashi-Osaka, Osaka 577-8502 (Japan); Satoh, Megumi; Tateishi, Takahiro; Nojiri, Tomoaki; Takeuchi, Daisuke; Kimura, Takayoshi [Department of Chemistry, School of Science and Engineering, Kinki University, Kowakae 3-4-1, Higashi-Osaka, Osaka 577-8502 (Japan)


    Highlights: Black-Right-Pointing-Pointer Effects of cyclodextrin on stability and conformation of lysozyme were clarified. Black-Right-Pointing-Pointer The CD influences the hydrophobic interaction of lysozyme by the inclusion. Black-Right-Pointing-Pointer The CD relatively destabilized the folded state by stabilizing the unfolded state. Black-Right-Pointing-Pointer The destabilization depends on the concentration and the substituent of CD. Black-Right-Pointing-Pointer The conformation of lysozyme was more spread at unfolded state by inclusion of CD. - Abstract: Effects of cyclic oligosaccharide cyclodextrin (CD) on stability and conformation of lysozyme were clarified thermodynamically and rheologically by DSC, viscosity, and circular dichroism measurements. The modified {beta}-CD relatively destabilized the folded state of lysozyme by stabilizing the unfolded state due to inclusion of hydrophobic part into the hydrophobic interior of CD. The order of higher destabilization effect was acetyl-{beta}-CD > methyl-{beta}-CD > hydroxypropyl-{beta}-CD. Apparent number of bound CD to unfolded state for methyl-, hydroxypropyl-, and acetyl-{beta}-CD is 6.7 {+-} 0.7, 4.2 {+-} 1.1, and 18.6 {+-} 4.3 and the binding constant is 5.5 {+-} 0.8, 6.7 {+-} 2.4, and 4.4 {+-} 1.2 L mol{sup -1}, respectively. The viscosity for unfolded state was increased with an increase in the each modified {beta}-CD concentration, suggesting that the inclusion of CD on a part of hydrophobic core at unfolded state leads to break the hydrophobic core, then lysozyme would be more spread structure. The substituent of CD can accelerate instability by directly breaking hydrogen bond and/or can restrain instability by increase in hydrophobic interaction. The fact that the each modified CDs has different destabilization effect shows a possibility to control the stability of protein by the substitution of CD.

  8. Adsorption of lysozyme on hyaluronic acid functionalized SBA-15 mesoporous silica: a possible bioadhesive depot system. (United States)

    Medda, Luca; Casula, Maria F; Monduzzi, Maura; Salis, Andrea


    Silica-based ordered mesoporous materials are very attractive matrices to prepare smart depot systems for several kinds of therapeutic agents. This work focuses on the well-known SBA-15 mesoporous silica and lysozyme, an antimicrobial protein. In order to improve the bioadhesion properties of SBA-15 particles, the effect of hyaluronic acid (HA) functionalization on lysozyme adsorption was investigated. SBA-15 samples having high (H-SBA) and low (L-SBA) levels of functionalization were analyzed during the three steps of the preparations: (1) introduction of the -NH2 groups to obtain the SBA-NH2 samples; (2) functionalization with HA to obtain the SBA-HA matrices; (3) adsorption of lysozyme. All silica matrices were characterized through N2-adsorption/desorption isotherms, small-angle X-ray scattering, transmission electron microscopy, thermogravimetric analysis, and Fourier transform infrared spectroscopy. The whole of the experimental data suggests that a high level of functionalization of the silica surface allows for a negligible lysozyme adsorption mainly due to unfavorable electrostatic interactions (H-SBA-NH2) or steric hindrance (H-SBA-HA). A low degree of functionalization of the silica surface brings about a very good performance toward lysozyme adsorption, being 71% (L-SBA-NH2) and 63% (L-SBA-HA) respectively, compared to that observed for original SBA-15. Finally, two different kinetic models--a "pseudo-second order" and a "intraparticle diffusion"--were compared to fit lysozyme adsorption data, the latter being more reliable than the former.

  9. Fluid of fused spheres as a model for protein solution

    Directory of Open Access Journals (Sweden)

    M. Kastelic


    Full Text Available In this work we examine thermodynamics of fluid with "molecules" represented by two fused hard spheres, decorated by the attractive square-well sites. Interactions between these sites are of short-range and cause association between the fused-sphere particles. The model can be used to study the non-spherical (or dimerized proteins in solution. Thermodynamic quantities of the system are calculated using a modification of Wertheim's thermodynamic perturbation theory and the results compared with new Monte Carlo simulations under isobaric-isothermal conditions. In particular, we are interested in the liquid-liquid phase separation in such systems. The model fluid serves to evaluate the effect of the shape of the molecules, changing from spherical to more elongated (two fused spheres ones. The results indicate that the effect of the non-spherical shape is to reduce the critical density and temperature. This finding is consistent with experimental observations for the antibodies of non-spherical shape.

  10. Preparation of cross-linked hen-egg white lysozyme crystals free of cracks (United States)

    Yan, Er-Kai; Lu, Qin-Qin; Zhang, Chen-Yan; Liu, Ya-Li; He, Jin; Chen, Da; Wang, Bo; Zhou, Ren-Bin; Wu, Ping; Yin, Da-Chuan


    Cross-linked protein crystals (CLPCs) are very useful materials in applications such as biosensors, catalysis, and X-ray crystallography. Hence, preparation of CLPCs is an important research direction. During the preparation of CLPCs, an often encountered problem is that cracks may appear in the crystals, which may finally lead to shattering of the crystals into small pieces and cause problem in practical applications. To avoid cross-link induced cracking, it is necessary to study the cracking phenomenon in the preparation process. In this paper, we present an investigation on how to avoid cracking during preparation of CLPCs. An orthogonal experiment was designed to study the phenomenon of cross-link induced cracking of hen-egg white lysozyme (HEWL) crystals against five parameters (temperature, solution pH, crystal growth time, glutaraldehyde concentration, and cross-linking time). The experimental results showed that, the solution pH and crystal growth time can significantly affect cross-link induced cracking. The possible mechanism was studied, and optimized conditions for obtaining crack-free CLPCs were obtained and experimentally verified. PMID:27703210

  11. 一种温敏性水凝胶复性体系:聚N-异丙基丙烯酰胺的制备及其在溶菌酶复性中的应用%A Temperature-sensitive Hydrogel Refolding System:Preparation of Poly(N-isopropyl acrylamide) and Its Application in Lysozyme Refolding

    Institute of Scientific and Technical Information of China (English)

    崔志芳; 关怡新; 姚善泾


    Temperature-sensitive hydrogel-poly(N-isopropyl acrylamide) (PNIPA) was prepared and applied to protein refolding. PNIPA gel disks and gel particles were synthesized by the solution polymerization and inverse suspension polymerization respectively. The swelling kinetics of the gels was also studied. With these prepared PNIPA gels, the model protein lysozyme was renatured. Within 24 h, PNIPA gel disks improved the yield of lysozyme activity by 49.3% from 3375.2 to 5038.8 With the addition of faster response PNIPA gel beads,the total lysozyme activity recovery was about 68.98% in 3h, as compared with 42.03% by simple batch dilution.The novel refolding system with PNIPA enables efficient refolding especially at high protein concentrations. Discussion about the mechanism revealed that when PNIPA gels were added into the refolding buffer, the hydrophobic interactions between denatured proteins and polymer gels could prevent the aggregation of refolding intermediates,thus enhanced the protein renaturation.

  12. Solvent-induced lysozyme gels: rheology, fractal analysis, and sol-gel kinetics. (United States)

    da Silva, Marcelo A; Arêas, Elizabeth P G


    In this work, the gelation kinetics and fractal character of lysozyme gel matrices developed in tetramethylurea (TMU)-water media were investigated. Gelation times were determined from the temporal crossover point between the storage, G', and loss, G'', moduli, as a function of the binary solvent composition and of protein concentration. The inverse dependence of the upper limit of the linear viscoelastic region (gamma0) on protein concentration indicate that the lysozyme gels belong to the "strong link" kind, a gel category where interparticle links are stronger than intraparticle ones. Lysozyme gel fractal dimensions (Df) were determined from the analysis of rheological data according to a scaling theory by Shih et al. [Phys. Rev. A 42 (1990) 4772-4779] and were found to be compatible with a diffusion-limited cluster-aggregation kinetics (DLCA) for lysozyme gels formed at the TMU mass fraction in the binary organic-aqueous solvent, wTMU=0.9, and with a reaction-limited cluster aggregation kinetics (RLCA) for wTMU in the 0.6< or =wTMU< or =0.8 range.

  13. Study of lysozyme mobility and binding free energy during adsorption on a graphene surface

    Energy Technology Data Exchange (ETDEWEB)

    Nakano, C. Masato [Flintridge Preparatory School, La Canada Flintridge, California 91011 (United States); Ma, Heng; Wei, Tao, E-mail: [Dan F. Smith Department of Chemical Engineering, Lamar University, Beaumont, Texas 77710 (United States)


    Understanding protein adsorption is a key to the development of biosensors and anti-biofouling materials. Hydration essentially controls the adsorption process on hydrophobic surfaces, but its effect is complicated by various factors. Here, we present an ideal model system to isolate hydration effects—lysozyme adsorption on a flat hydrophobic graphene surface. Our all-atom molecular dynamics and molecular-mechanics/Poisson-Boltzmann surface area computation study reveal that lysozyme on graphene displays much larger diffusivity than in bulk water. Protein's hydration free energy within the first hydration shell is dominated by the protein-water electrostatic interactions and acts as an energy barrier for protein adsorption. On the other hand, the surface tension, especially that from the hydrophobic graphene, can effectively weaken the barrier to promote adsorption.

  14. 甲基纤维素湿敏水凝胶中溶菌酶微球性质的体外评价%In vitro Evaluation of Lysozyme-loaded Microspheres in Thermosensitive Methylcellulose-based Hydrogel

    Institute of Scientific and Technical Information of China (English)

    林莹; 孙佳丽; 蒋国强; 昝佳; 丁富新


    Long-term injectable microspheres have some inherent disadvantages such as migration of microspheres from the original site and the burst effect. In order to avoid these problems, microsphere-loaded thermosensitive hydrogel system was designed and expected to achieve a zero-order release of biomolecular drugs in relative high initial drug loadings. Lysozyme, an antibacterial protein usually used to reduce prosthetic valve endocarditis,was selected as the model drug. Poly (DL-lactide-co-glycolide) (PLGA) microspheres, prepared by solvent evaporation method, were employed to encapsulate lysozyme and dispersed into thermosensitive pre-gel solution containing methylcellulose (MC), polyethylene glycol (PEG), sodium citrate (SC), and sodium alginate (SA). The mixture could act as a drug reservoir by performing sol-gel transition rapidly if the temperature was raised from room temperature to 37℃. The in vitro release results showed that the burst effect was avoided due to strengthening of diffusion resistance in the gel. The formulation was able to deliver lysozyme for over 30 days in a nearly zero-order release profile with a rate of 32.8μg·d-1 which exhibits its remarkable potential for effective application in long-term drug delivery.

  15. Cloning and molecular characterization of two invertebrate-type lysozymes from Anopheles gambiae


    Paskewitz, S. M.; Li, B.; Kajla, M. K.


    We sequenced and characterized two novel invertebrate-type lysozymes from the mosquito Anopheles gambiae. Alignment and phylogenetic analysis of these and a number of related insect proteins identified through bioinformatics strategies showed a high degree of conservation of this protein family throughout the Class Insecta. Expression profiles were examined for the two mosquito genes through semiquantitative and real-time PCR analysis. Lys i-1 transcripts were found in adult females in the fa...

  16. Physicochemical characterization and sorption behavior of Mg-Ca-Al (NO{sub 3}) hydrotalcite-like compounds toward removal of fluoride from protein solutions

    Energy Technology Data Exchange (ETDEWEB)

    Lv, Tengfei, E-mail: [Department of Chemistry, Dalian University of Technology, Dalian 116023 (China); Ma, Wei, E-mail: [Department of Chemistry, Dalian University of Technology, Dalian 116023 (China); Xin, Gang; Wang, Ren; Xu, Jun [Department of Chemistry, Dalian University of Technology, Dalian 116023 (China); Liu, Dongmei; Liu, Fujun; Pan, Decong [Dalian Ocean Fishery Group of Corporations, Dalian 116023 (China)


    Highlights: Black-Right-Pointing-Pointer Synthesize MgCaAlNO{sub 3} hydrotalcite-like compounds (HTlcs) with different Ca content. Black-Right-Pointing-Pointer The protein systems were evaluated for the removal of fluoride by the HTlcs. Black-Right-Pointing-Pointer Mg{sub 2.5}Ca{sub 0.5}Al-HTlc performs the best fluoride binding ability in protein solutions. Black-Right-Pointing-Pointer Extremely low losses of proteins were observed during adsorption process. Black-Right-Pointing-Pointer Antarctic krill wastewater study confirmed the practicality of the adsorbent. - Abstract: The present study explores the potential of Mg-Ca-Al (NO{sub 3}) hydrotalcite-like compounds (MgCaAlNO{sub 3}-HTlcs) for the removal of fluoride from protein solutions. In this study, the Mg{sub 3-x}Ca{sub x}AlNO{sub 3}-HTlcs (x = 0-3, x is the mol.% of Ca) were synthesized and characterized by SEM, XRD, FTIR, BET, ICP-AES and pHzpc analysis. The sorption experiments were conducted in protein systems of bovine serum albumin (BSA) and lysozyme (LSZ). The batch experiment results showed that the NO{sub 3}-HTlc with Mg/Ca/Al molar ratio of 2.5/0.5/1 had remarkable fluoride sorption ability with maximum sorption capacities of 82.35 mg/g and 72.69 mg/g at pH 5.0 and 40 Degree-Sign C in BSA and LSZ system, respectively. Moreover, the loss of BSA of 0.71% was low and there was no loss of LSZ. It was evident that the Mg{sub 2.5}Ca{sub 0.5}AlNO{sub 3}-HTlc could selectively adsorb fluoride from protein solutions. The equilibrium sorption data fitted well to the Langmuir model and the kinetic data conformed to the pseudo-second-order model. Thermodynamic parameters ({Delta}G Degree-Sign , {Delta}H Degree-Sign and {Delta}S Degree-Sign ) were evaluated and revealed that the sorption process was spontaneous and endothermic in nature. Furthermore, the results from Antarctic krill processing wastewater study confirmed the feasibility and practicality of the Mg{sub 2.5}Ca{sub 0.5}AlNO{sub 3}-HTlc for fluoride

  17. Protein adsorption and separation on amphoteric chitosan/carboxymethylcellulose membranes. (United States)

    Feng, Zhicheng; Shao, Zhengzhong; Yao, Jinrong; Chen, Xin


    This article reported the preparation of an amphoteric natural polymeric membrane-macroporous chitosan (CS)/carboxymethylcellulose (CMC) blend membrane and the utilization of such a membrane on the membrane chromatography for bioseparation. The membranes were prepared by solution blending of CS and CMC solution, and using silica particles as porogen. Both glutaraldehyde and epichlorohydrin were used as crosslinking agent to increase its chemical stability in aqueous solution. Such a natural polymeric membrane can be served as an amphoteric membrane because of the amino group on CS and the carboxymethyl group on CMC, in which the surface charge can be changed with the environmental pH. Ovalbumin (pI = 4.6) and lysozyme (pI = 11) were selected as model proteins. These two proteins adsorption on different CS/CMC blend membranes with different initial protein concentrations at different pH values were investigated in batch systems. The results indicated that the maximum adsorption for lysozyme and ovalbumin was at pH 9.2 and 4.8 respectively, and the adsorption capacity on the membrane both increased with the increase of initial protein concentration. Though the adsorption mechanism of lysozyme and ovalbumin was found not the same, the maximum adsorption capacity of two proteins on the membranes was quite similar (about 250 mg/g). Moreover, the desorption ratio of both proteins was found to be more than 90% that implied CS/CMC blend membrane could separate proteins by adsorption-desorption process. Finally, both lysozyme and ovalbumin were successfully separated from their binary mixture only by adjusting the pH of the feed and the desorption solution.

  18. The influence of lysozyme on mannitol polymorphism in freeze-dried and spray-dried formulations depends on the selection of the drying process. (United States)

    Grohganz, Holger; Lee, Yan-Ying; Rantanen, Jukka; Yang, Mingshi


    Freeze-drying and spray-drying are often applied drying techniques for biopharmaceutical formulations. The formation of different solid forms upon drying is often dependent on the complex interplay between excipient selection and process parameters. The purpose of this study was to investigate the influence of the chosen drying method on the solid state form. Mannitol-lysozyme solutions of 20mg/mL, with the amount of lysozyme varying between 2.5% and 50% (w/w) of total solid content, were freeze-dried and spray-dried, respectively. The resulting solid state of mannitol was analysed by near-infrared spectroscopy in combination with multivariate analysis and further, results were verified with X-ray powder diffraction. It was seen that the prevalence of the mannitol polymorphic form shifted from β-mannitol to δ-mannitol with increasing protein concentration in freeze-dried formulations. In spray-dried formulations an increase in protein concentration resulted in a shift from β-mannitol to α-mannitol. An increase in final drying temperature of the freeze-drying process towards the temperature of the spray-drying process did not lead to significant changes. It can thus be concluded that it is the drying process in itself, rather than the temperature, that leads to the observed solid state changes.

  19. A novel bottom-up process to produce nanoparticles containing protein and peptide for suspension in hydrofluoroalkane propellants. (United States)

    Tan, Yinhe; Yang, Zhiwen; Peng, Xinsheng; Xin, Feng; Xu, Yuehong; Feng, Min; Zhao, Chunshun; Hu, Haiyan; Wu, Chuanbin


    To overcome the disadvantages of microemulsion and nanoprecipitation methods to produce protein-containing nanoparticles, a novel bottom-up process was developed to produce nanoparticles containing the model protein lysozyme. The nanoparticles were generated by freeze-drying a solution of lysozyme, lecithin and lactose in tert-butyl alcohol (TBA)/water co-solvent system and washing off excess lecithin in lyophilizate by centrifugation. Formulation parameters such as lecithin concentration in organic phase, water content in TBA/water co-solvent, and lactose concentration in water were optimized so as to obtain desired nanoparticles with retention of the bioactivity of lysozyme. Based on the results, 24.0% (w/v) of lecithin, 37.5% (v/v) of water content, and 0.56% (w/v) of lactose concentration were selected to generate spherical nanoparticles with approximately 200 nm in mean size, 0.1 in polydispersity index (PI), and 99% retained bioactivity of lysozyme. These nanoparticles rinsed with ethanol containing dipalmitoylphosphatidylcholine (DPPC), Span 85 or oleic acid (3%, w/v) could readily be dispersed in HFA 134a to form a stable suspension with good redispersibility and 98% retained bioactivity of lysozyme. The study indicates there is a potential to produce pressed metered dose inhaler (pMDI) formulations containing therapeutic protein and peptide nanoparticles.

  20. Lysozyme adsorption on the colloidal chromium(III) oxide surface: Its impact on the system stability

    Energy Technology Data Exchange (ETDEWEB)

    Szewczuk-Karpisz, Katarzyna, E-mail:; Wiśniewska, Małgorzata; Myśliwiec, Dawid


    Highlights: • Lysozyme adsorption mechanism on the chromium(III) oxide surface was determined. • Surface charge density as well as zeta potential of Cr{sub 2}O{sub 3} particles were measured. • Turbidimetric method was used to estimate the suspension stability. • Depending on the pH value, lysozyme increases or decreases the system stability. - Abstract: This paper describes the lysozyme (LSZ) presence effect on the chromium(III) oxide (Cr{sub 2}O{sub 3}) suspension stability. First, the electrokinetic properties of the examined system, i.e. surface charge density and zeta potential of solid particles in the absence and presence of LSZ, were determined. The lysozyme addition reduces the metal oxide surface charge, which may be related to the interaction of the LSZ protonated amino groups with the adsorbent surface moieties. The LSZ macromolecules undergo adsorption on the Cr{sub 2}O{sub 3} surface only under electrostatic attraction. At the LSZ concentrations above 50 ppm the macromolecules cover completely the particle surface, which is evidenced by the observed zeta potential values. The LSZ influence on the Cr{sub 2}O{sub 3} suspension stability depends on the solution pH value. At pH 3, 4.6 and 7.6, the LSZ addition improves the system stability. In turn, at pH 9 it is associated with the slight suspension destabilization.

  1. Reduction of protein adsorption on silica and polysulfone surfaces coated with complex coacervate core micelles with poly(vinyl alcohol) as a neutral brush forming block

    NARCIS (Netherlands)

    Brzozowska, A. M.; Zhang, Q.; de Keizer, A.; Norde, W.; Stuart, M. A. Cohen


    We have studied the formation and stability of complex coacervate core micelles (C3Ms) in solution, and the influence of C3M coatings on the adsorption of the proteins beta-lactoglobulin (beta-lac), bovine serum albumin (BSA). and lysozyme (Lsz) on silica and polysulfone surfaces. The C3M5 consist o

  2. 溶菌酶在酱油中的抑菌效果研究%Studies of lysozyme on the inhibiting efficiency of the microorganisms in soy sauce

    Institute of Scientific and Technical Information of China (English)



    Lysozyme is a protein molecule that is comprised of a polypeptide chain. It has good thermal stability in the acid solutions, and words well in killing Gram - positive bacteria. So it is a better and natural preservative than the others which is used in soy sauce. The effect of lysozyme on inhibiting microorganisms in soy sauce was studied in this research. The results showed that; (1) the best dosage of lysozyme was 150 mg/kg in soy sauce, which could inhibite microorganisms well, and kill the microorganisms slowly. Then the antiseptic effect of lysozyme was better 0. 1% sodium benzoate. (2) Lysozyme worked better in the soy sauce that has high salt content than the low one, and the total plate count of the soy sauce decreased much more obviously. (3) Pasteurisation affected the lysozyme activity little. (4) Lysozyme inhibited microorganisms well in soy sauce, in which the total plate count was no more than 10000cfu/ml. and increased slowly. The product quality of soy sauce was stable during the shelf - life.%溶菌酶为多肽链组成的蛋白质分子,酶活在酸性条件下具有良好的热稳定性,且对革兰氏阳性细菌具有专一的抑制杀灭作用,是成品酱油的一种良好的天然抑菌防腐剂.本文研究了溶菌酶在酱油中的抑菌防腐效果.结果表明:(1)在酱油中,溶菌酶的抑菌防腐效果理想,且具有缓慢杀菌的功能,其最佳添加量为150 mg/kg,抑菌防腐效果比0.1%苯甲酸钠要好;(2)在高盐分含量酱油中的抑菌防腐效果比低盐分含量酱油要好,添加溶菌酶后菌落总数下降趋势更加明显;(3)酱油的巴氏灭菌操作对溶菌酶的活性无显著影响;(4)在起始菌落总数为10000 cfu/ml或以下的酱油中使用溶菌酶能使酱油具有较好的抑菌防腐效果,在货架期内菌落总数增长缓慢,产品质量稳定.

  3. Lysozyme refolding at high concentration by dilution and size-exclusion chromatography

    Institute of Scientific and Technical Information of China (English)


    This study of renaturation by dilution and size exclusion chromatography (SEC) addition of urea to improve yield as well as the initial and final protein concentrations showed that although urea decreased the rate of lysozyme refolding, it could suppress protein aggregation to sustain the pathway of correct refolding at high protein concentration; and that there existed an optimum urea concentration in renaturation buffer. Under the above conditions, lysozyme was successfully refolded from initial concentration of up to 40 mg/mL by dilution and 100 mg/mL by SEC, with the yield of the former being more than 40% and that of the latter being 34.8%. Especially, under the condition of 30 min interval time, i.e. τ>2(tR2-tR1), the efficiency was increased by 25% and the renaturation buffer could be recycled for SEC refolding in continuous operation of downstream process.

  4. Quinopeptide formation associated with the disruptive effect of epigallocatechin-gallate on lysozyme fibrils. (United States)

    Cao, Na; Zhang, Yu-Jie; Feng, Shuang; Zeng, Cheng-Ming


    Numerous studies demonstrate that natural polyphenols can inhibit amyloid formation and disrupt preformed amyloid fibrils. In the present study, the fibril-disruptive effects of epigallocatechin-3-gallate (EGCG) were examined using lysozyme as a model protein. The results indicated that EGCG dose dependently inhibited lysozyme fibrillation and modified the peptide chains with quinonoid moieties under acidic conditions, as measured by ThT fluorescence, transmission electron microscopy, and an NBT-staining assay. Moreover, EGCG transformed the preformed lysozyme fibrils to amorphous aggregates through quinopeptide formation. The thiol blocker, N-ethylmaleimide, inhibited the disruptive effect of EGCG on preformed fibrils, suggesting that thiol groups are the binding sites for EGCG. We propose that the formation of quinone intermediates via oxidation and subsequent binding to lysozyme chains are the main processes driving the inhibition of amyloid formation and disruption of preformed fibrils by EGCG. The information presented in this study may provide fresh insight into the link between the antioxidant capacity and anti-amyloid activity of polyphenols. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Behavior of lysozyme adsorbed onto biological liquid crystal lipid monolayer at the air/water interface (United States)

    Lu, Xiaolong; Shi, Ruixin; Hao, Changchun; Chen, Huan; Zhang, Lei; Li, Junhua; Xu, Guoqing; Sun, Runguang


    The interaction between proteins and lipids is one of the basic problems of modern biochemistry and biophysics. The purpose of this study is to compare the penetration degree of lysozyme into 1,2-diapalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethano-lamine (DPPE) by analyzing the data of surface pressure-area (π-A) isotherms and surface pressure-time (π-T) curves. Lysozyme can penetrate into both DPPC and DPPE monolayers because of the increase of surface pressure at an initial pressure of 15 mN/m. However, the changes of DPPE are larger than DPPC, indicating stronger interaction of lysozyme with DPPE than DPPC. The reason may be due to the different head groups and phase state of DPPC and DPPE monolayers at the surface pressure of 15 mN/m. Atomic force microscopy reveals that lysozyme was absorbed by DPPC and DPPE monolayers, which leads to self-aggregation and self-assembly, forming irregular multimers and conical multimeric. Through analysis, we think that the process of polymer formation is similar to the aggregation mechanism of amyloid fibers. Project supported by the National Natural Science Foundation of China (Grant Nos. 21402114 and 11544009), the Natural Science Basic Research Plan in Shaanxi Province of China (Grant No. 2016JM2010), the Fundamental Research Funds for the Central Universities of China (Grant No. GK201603026), and the National University Science and Technology Innovation Project of China (Grant No. 201610718013).

  6. Basal Secretion of Lysozyme from Human Airways in Vitro

    Directory of Open Access Journals (Sweden)

    Patricia Roger


    Full Text Available The aim of this study was to examine the basal release of lysozyme from isolated human lung tissues. Measurements of lysozyme in the fluids derived from lung preparations were performed using a rate-of-lysis assay subsequent to acidification of the biological samples. Lysozyme released from bronchial preparations into fluids was greater than that observed for parenchymal tissues. The lysozyme quantities detected in bronchial fluids were not modified by removal of the surface epithelium. Furthermore, the quantities of lysozyme in bronchial fluids was correlated with the size of the bronchial preparations. These results suggest that the lysozyme was principally secreted by the human bronchi (submucosal layer rather than by parenchyma tissues and that a greater release was observed in the proximal airways.

  7. Oxalic acid complexes: Promising draw solutes for forward osmosis (FO) in protein enrichment

    KAUST Repository

    Ge, Qingchun


    Highly soluble oxalic acid complexes (OACs) were synthesized through a one-pot reaction. The OACs exhibit excellent performance as draw solutes in FO processes with high water fluxes and negligible reverse solute fluxes. Efficient protein enrichment was achieved. The diluted OACs can be recycled via nanofiltration and are promising as draw solutes.

  8. Crystallization of lysozyme with (R)-, (S)- and (RS)-2-methyl-2, 4-pentanediol

    Energy Technology Data Exchange (ETDEWEB)

    Stauber, Mark [Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312 (United States); Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312 (United States); Jakoncic, Jean [Brookhaven National Laboratory, Building 725D, Upton, NY 11973-5000 (United States); Berger, Jacob; Karp, Jerome M.; Axelbaum, Ariel; Sastow, Dahniel [Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312 (United States); Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312 (United States); Buldyrev, Sergey V. [Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312 (United States); Hrnjez, Bruce J. [Collegiate School, 260 West 78th Street, New York, NY 10024-6559 (United States); Asherie, Neer, E-mail: [Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312 (United States); Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312 (United States)


    Crystallization of lysozyme with (R)-2-methyl-2, 4-pentanediol produces more ordered crystals and a higher resolution protein structure than crystallization with (S)-2-methyl-2, 4-pentanediol. The results suggest that chiral interactions with chiral additives are important in protein crystal formation. Chiral control of crystallization has ample precedent in the small-molecule world, but relatively little is known about the role of chirality in protein crystallization. In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2, 4-pentanediol (MPD) separately using the R and S enantiomers as well as with a racemic RS mixture. Crystals grown with (R)-MPD had the most order and produced the highest resolution protein structures. This result is consistent with the observation that in the crystals grown with (R)-MPD and (RS)-MPD the crystal contacts are made by (R)-MPD, demonstrating that there is preferential interaction between lysozyme and this enantiomer. These findings suggest that chiral interactions are important in protein crystallization.

  9. Carboxymethyl chitosan-poly(amidoamine) dendrimer core-shell nanoparticles for intracellular lysozyme delivery. (United States)

    Zhang, Xiaoyang; Zhao, Jun; Wen, Yan; Zhu, Chuanshun; Yang, Jun; Yao, Fanglian


    Intracellular delivery of native, active proteins is challenging due to the fragility of most proteins. Herein, a novel polymer/protein polyion complex (PIC) nanoparticle with core-shell structure was prepared. Carboxymethyl chitosan-grafted-terminal carboxyl group-poly(amidoamine) (CM-chitosan-PAMAM) dendrimers were synthesized by amidation and saponification reactions. (1)H NMR was used to characterize CM-chitosan-PAMAM dendrimers. The TEM images and results of lysozyme loading efficiency indicated that CM-chitosan-PAMAM dendrimers could self-assemble into core-shell nanoparticles, and lysozyme was efficiently encapsulated inside the core of CM-chitosan-PAMAM dendrimer nanoparticles. Activity of lysozyme was completely inhibited by CM-chitosan-PAMAM Dendrimers at physiological pH, whereas it was released into the medium and exhibited a significant enzymatic activity in an acidic intracellular environment. Moreover, the CM-chitosan-PAMAM dendrimer nanoparticles did not exhibit significant cytotoxicity in the range of concentrations below 3.16 mg/ml. The results indicated that these CM-chitosan-PAMAM dendrimers have excellent properties as highly potent and non-toxic intracellular protein carriers, which would create opportunities for novel applications in protein delivery.

  10. A computational solution to analyze hydrophobic characteristics in protein chains (United States)

    Soares, Marilia Amável Gomes; Azevedo, Alexandre; Missailidis, Sotiris; Silva, Dilson


    This paper presents a program developed to facilitate calculations of the total or partial hidrophobicity value of polypeptides and proteins chains. It was built using the Fortran 77 language and performs additional functions, determining the total free energy and the electromotive force of an amino acid in a protein. These values were then used to estimate the average hydrophobicity of the protein or fragment sequence.

  11. 动物源溶菌酶研究进展%Advance in Studies of Animal-borne Lysozyme

    Institute of Scientific and Technical Information of China (English)

    张鹏; 江明锋; 王永


    动物源溶菌酶是一种动物体内广泛存在的酶类,它可以水解细菌细胞壁肽聚糖中的β-1,4糖苷键,具有消化分解细菌、抑制外源微生物生长、增强机体免疫力的作用.目前溶菌酶已被用作研究蛋白功能、性质以及分子进化的模型.首先介绍了溶菌酶及其分子的晶体结构,溶菌酶基因及其蛋白研究进展,其次介绍了动物源溶菌酶的功能,包括溶菌酶生物学功能和重组蛋白功能活性,重点介绍了溶菌酶基因在转基因工程中的应用研究,最后对动物源溶菌酶研究进行了展望.研究动物源溶菌酶对于基础科学,并应用其转变成现实生产力具有重要的指导意义.%Lysozyme is a kind of muramidases that widespread in the animal in vivo, and can catalyze the hydrolysis of β-1, 4-glycosidic bonds between the N-acetylglucosamine and N-acetylmuramic acid in the peptidoglycan layer of bacterial cell walls. It has functions of the digestion and decomposition of bacteria, and inhibition of exogenous microbial growth, and enhancing immunity; and lysozyme has been a model protein for research in the function and the nature of the enzyme, and molecular evolution. Firstly, the lysozyme and its crystal structure and the advance in studies of lysozyme gene and its protein were introduced. Secondly, the functions of animal-borne lysozyme, including the biological functions of lysozyme and functional activities of recombinant proteins were introduced too,then the applied researches in lysozyme gene in transgenic engineering were focused on. Finally, the perspectives of animal-borne lysozyme were suggested. It' s very significant to research the animal-borne lysozyme because it' s helpful to understand the basic knowledge and to use it in the production.

  12. Solution Properties of Soy Protein in DMSO/Urea Solvent System

    Institute of Scientific and Technical Information of China (English)

    XIAO Ru; YIN Duan; JIN Xin; SUN Gang


    Solution properties of 7S globulins (7S), 11S globulins (11S) and soy protein isolates (SPI) in dimethylsulfoxide ( DMSO )/urea solvent system were studied by intrinsic viscosity and particle size distributions.The results showed that the existence of urea was the main reason for the denaturation and solubility of soy protein in the system, and the effects were more obvious with increasing of urea concentration in solutions.Suitably dissolution temperature and time contributed to the solubility of soy proteins.

  13. Protein Condensation (United States)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.


    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  14. Hydrodynamic multibead modeling: problems, pitfalls, and solutions. 2. Proteins. (United States)

    Zipper, Peter; Durchschlag, Helmut


    Hydrodynamic models of proteins have been generated by recourse to crystallographic data and applying a filling model strategy in order to predict both hydrodynamic and scattering parameters. The design of accurate protein models retaining the majority of the molecule peculiarities requires usage of many beads and consideration of many serious problems. Applying the expertise obtained with ellipsoid models and pilot tests on proteins, we succeeded in constructing precise models for several anhydrous and hydrated proteins of different shape, size, and complexity. The models constructed consist of many beads (up to about 11,000) for the protein constituents (atoms, amino acid residues, groups) and preferentially bound water molecules. While in the case of small proteins, parameter predictions are straightforward, computations for giant proteins necessitate drastic reductions of the number of initially available beads. Among several auxiliary programs, our advanced hydration programs, HYDCRYST and HYDMODEL, and modified versions of García de la Torre's program HYDRO were successfully employed. This allowed the generation of realistic protein models by imaging details of their fine structure and enabled the prediction of reliable molecular parameters including intrinsic viscosities. The appearance of the models and the agreement of molecular properties and distance distribution functions p(r) of unreduced and reduced models can be used for a meticulous inspection of the data obtained.

  15. The balance of flexibility and rigidity in the active site residues of hen egg white lysozyme

    Institute of Scientific and Technical Information of China (English)

    Qi Jian-Xun; Jiang Fan


    The crystallographic temperature factors (B factor) of individual atoms contain important information about the thermal motion of the atoms in a macromolecule. Previously the theory of flexibility of active site has been established based on the observation that the enzyme activity is sensitive to low concentration denaturing agents. It has been found that the loss of enzyme activity occurs well before the disruption of the three-dimensional structural scaffold of the enzyme. To test the theory of conformational flexibility of enzyme active site, crystal structures were perturbed by soaking in low concentration guanidine hydrochloride solutions. It was found that many lysozyme crystals tested could still diffract until the concentration of guanidine hydrochloride reached 3 M. It was also found that the B factors averaged over individually collected data sets were more accurate. Thus it suggested that accurate measurement of crystal temperature factors could be achieved for medium-high or even medium resolution crystals by averaging over multiple data sets. Furthermore, we found that the correctly predicted active sites included not only the more flexible residues, but also some more rigid residues. Both the flexible and the rigid residues in the active site played an important role in forming the active site residue network, covering the majority of the substrate binding residues. Therefore, this experimental prediction method may be useful for characterizing the binding site and the function of a protein, such as drug targeting.

  16. Small-angle neutron scattering study of structural evolution of different phases in protein solution

    Indian Academy of Sciences (India)

    V K Aswal; S Chodankar; J Kohlbrecher; R Vavrin; A G Wagh


    Small-angle neutron scattering (SANS) has been used to study the structural evolution of different phases in protein solution leading to crystallization, denaturation and gelation. The protein solution under crystallization mostly consists of monomers and dimers, and higher-mers are not observed as they are perhaps formed in very small numbers. The onset and the rate of crystallization strongly depend on the salt concentration. Protein denaturation on addition of surfactant occurs due to the formation of micelle-like clusters along the unfolded polypeptide chains of the protein. The structure of such protein{surfactant complex is found to be independent of the size of the micelles in their pure surfactant solutions. The structure of temperature-induced protein gels shows a fractal structure. Rheology of these gels shows a strong dependence on varying pH or protein concentration, whereas the structure of such gels is found to be similar.

  17. Terahertz response of dipolar impurities in polar liquids: On anomalous dielectric absorption of protein solutions

    CERN Document Server

    Matyushov, D V


    A theory of radiation absorption by dielectric mixtures is presented. The coarse-grained formulation is based on the wavevector-dependent correlation functions of molecular dipoles of the host polar liquid and a density-density structure factor of the positions of the solutes. A nonlinear dependence of the absorption coefficient on the solute concentration is predicted and originates from the mutual polarization of the liquid surrounding the solutes by the collective field of the solute dipoles aligned along the radiation field. The theory is applied to terahertz absorption of hydrated saccharides and proteins. While the theory gives an excellent account of the observations for saccharides without additional assumptions and fitting parameters, experimental absorption coefficient of protein solutions significantly exceeds theoretical calculations within standard dielectric models and shows a peak against the protein concentration. A substantial polarization of protein's hydration shell is required to explain t...

  18. The solution structure of an anti-CRISPR protein (United States)

    Maxwell, Karen L.; Garcia, Bianca; Bondy-Denomy, Joseph; Bona, Diane; Hidalgo-Reyes, Yurima; Davidson, Alan R.


    Bacterial CRISPR–Cas adaptive immune systems use small guide RNAs to protect against phage infection and invasion by foreign genetic elements. We previously demonstrated that a group of Pseudomonas aeruginosa phages encode anti-CRISPR proteins that inactivate the type I-F and I-E CRISPR–Cas systems using distinct mechanisms. Here, we present the three-dimensional structure of an anti-CRISPR protein and map a functional surface that is critical for its potent inhibitory activity. The interaction of the anti-CRISPR protein with the CRISPR–Cas complex through this functional surface is proposed to prevent the binding of target DNA. PMID:27725669

  19. Complexation between dodecyl sulfate surfactant and zein protein in solution. (United States)

    Ruso, Juan M; Deo, Namita; Somasundaran, P


    Interactions between sodium dodecyl sulfate and zein protein, a model system for the understanding of the effect of surfactants on skin, were investigated using a range of techniques involving UV-vis spectroscopy, TOC (total organic carbon analysis), electrophoresis, and static and dynamic light scattering. Zein protein was solubilized by SDS. The adsorption of SDS onto insoluble protein fraction caused the zeta potential of the complex to become more negative. From these values, we calculated the Gibbs energy of absorption, which decreases when the SDS concentration is raised. Finally the structure of the complex, based on the analysis by static and dynamic light scattering, is proposed to be rod like.

  20. High-performance computational solutions in protein bioinformatics

    CERN Document Server

    Mrozek, Dariusz


    Recent developments in computer science enable algorithms previously perceived as too time-consuming to now be efficiently used for applications in bioinformatics and life sciences. This work focuses on proteins and their structures, protein structure similarity searching at main representation levels and various techniques that can be used to accelerate similarity searches. Divided into four parts, the first part provides a formal model of 3D protein structures for functional genomics, comparative bioinformatics and molecular modeling. The second part focuses on the use of multithreading for

  1. Mechanistic studies on the release of lysozyme from twin-screw extruded lipid implants. (United States)

    Sax, Gerhard; Winter, Gerhard


    The influence of lipid melting on the in-vitro release of lysozyme from twin-screw extruded lipid implants was investigated. Triglyceride based implants were prepared by admixing of glycerol tristearin and various low melting lipids and subsequent twin-screw extrusion (tsc-extrusion) of these mixtures at moderate temperatures. Lysozyme was embedded as model protein and PEG 4000 or PEG 6000 was used as pore-forming excipient. By decreasing the amount of pore-forming agent from 40% to 0% lysozyme release became more sustained and the release kinetics changed from a matrix-type release profile to a linear release profile. Differential scanning calorimetry, X-ray diffraction and scanning electron microscopy measurements showed a change in implant structure upon long-term release (240 days) at 37 °C which was explained by partial matrix melting. In addition, partial melting of the implants was found to facilitate complete drug release at 37 °C whereas at 20 °C without partial melting 20% to 90% of the incorporated protein remained trapped in the implant matrix. In conclusion, partial melting of the implants during in-vitro release was found to be a major factor for the control of protein release from extruded implants and can be useful to trigger release, achieve in-vivo biodegradability and complete long-term protein release.

  2. Do protein crystals nucleate within dense liquid clusters? (United States)

    Maes, Dominique; Vorontsova, Maria A; Potenza, Marco A C; Sanvito, Tiziano; Sleutel, Mike; Giglio, Marzio; Vekilov, Peter G


    Protein-dense liquid clusters are regions of high protein concentration that have been observed in solutions of several proteins. The typical cluster size varies from several tens to several hundreds of nanometres and their volume fraction remains below 10(-3) of the solution. According to the two-step mechanism of nucleation, the protein-rich clusters serve as locations for and precursors to the nucleation of protein crystals. While the two-step mechanism explained several unusual features of protein crystal nucleation kinetics, a direct observation of its validity for protein crystals has been lacking. Here, two independent observations of crystal nucleation with the proteins lysozyme and glucose isomerase are discussed. Firstly, the evolutions of the protein-rich clusters and nucleating crystals were characterized simultaneously by dynamic light scattering (DLS) and confocal depolarized dynamic light scattering (cDDLS), respectively. It is demonstrated that protein crystals appear following a significant delay after cluster formation. The cDDLS correlation functions follow a Gaussian decay, indicative of nondiffusive motion. A possible explanation is that the crystals are contained inside large clusters and are driven by the elasticity of the cluster surface. Secondly, depolarized oblique illumination dark-field microscopy reveals the evolution from liquid clusters without crystals to newly nucleated crystals contained in the clusters to grown crystals freely diffusing in the solution. Collectively, the observations indicate that the protein-rich clusters in lysozyme and glucose isomerase solutions are locations for crystal nucleation.

  3. Resistance screening essay of wine lactic acid bacteria on lysozyme: efficacy of lysozyme in unclarified grape musts. (United States)

    Delfini, Claudio; Cersosimo, Manuela; Del Prete, Vincenzo; Strano, Morela; Gaetano, Giuseppe; Pagliara, Adolfo; Ambrò, Stefano


    In wine making, the bacteriolytic activity of lysozyme has primarily been used to control the malolactic fermentation in wines. The use of lysozyme in musts before settling and the beginning of the alcoholic fermentation to inhibit the growth of lactic acid bacteria could be very beneficial. In a resistance test carried out in MT/b broth, lysozyme had greater antimicrobial activity toward Oenococcus oeni than Lactobacillus species. Several strains of wine bacteria belonging to Oenococcus proved sensitive to the bacteriolytic activity of lysozyme at low concentrations in both synthetic medium (MT/b) (50 mg/L), white must, or red must made with or without the skins (100 mg/L). Lactobacillus and Pediococcus strains survived at lysozyme concentrations of 200-500 and 500 mg/L, respectively, in MT/b and musts. Suspended solids in unclarified musts may strongly bind to lysozyme thereby causing its removal by filtration or centrifugation. One hour after lysozyme was added to musts, it was quantified by HPLC and found after centrifugation to be 40-50% and only 10% in musts made with or without the skins, respectively. Although appreciable amounts of lysozyme were bound to wine components, this did not appear to be a serious hindrance to lysozyme activity.

  4. Direct biomolecule binding on nonfouling surfaces via newly discovered supramolecular self-assembly of lysozyme under physiological conditions. (United States)

    Yang, Peng


    When lysozyme is dissolved in a neutral HEPES buffer solution (pH = 7.4) with 0.001-0.050 M TCEP added, a fast phase transition process occurs and the resulting novel fiber-like hierarchical supramolecular assemblies made by primary spherical-particle aggregation can function as a "superglue" that binds strongly and quickly onto non-fouling coatings. This binding is highly selective towards lysozyme, and excludes synthetic, chemical/physical activation/deactivation (blocking) steps. By using biotinylated lysozyme, such a phase transition quickly creates a perfect biotinylated surface on non-fouling surfaces for avidin binding, showing great potential for the development of low-cost and practical biochips.

  5. Crystallization of Chicken Egg-White Lysozyme from Ammonium Sulfate (United States)

    Forsythe, Elizabeth L.; Snell, Edward H.; Pusey, Marc L.


    Chicken egg-white lysozyme was crystallized from ammonium sulfate over the pH range 4.0-7.8, with protein concentrations from 100 to 150 mg/ml. Crystals were obtained by vapor-diffusion or batch-crystallization methods. The protein crystallized in two morphologies with an apparent morphology dependence on temperature and protein concentration. In general, tetragonal crystals could be grown by lowering the protein concentration or temperature. Increasing the temperature or protein concentration resulted in the growth of orthorhombic crystals. Representative crystals of each morphology were selected for X-ray analysis. The tetragonal crystals belonged to the P4(sub 3)2(sub 1)2 space group with crystals grown at ph 4.4 having unit-cell dimensions of a = b = 78.7 1, c=38.6 A and diffracting to beyond 2.0 A. The orthorhombic crystals, grown at pH 4.8, were of space group P2(sub 1)2(sub 1)2 and had unit-cell dimensions of a = 30.51, b = 56.51 and c = 73.62 A.

  6. Complex Formation Between Lysozyme and Stabilized Micelles with a Mixed Poly(ethylene oxide)/Poly(acrylic acid) Shell. (United States)

    Karayianni, Maria; Gancheva, Valeria; Pispas, Stergios; Petrov, Petar


    The electrostatic complexation between lysozyme and stabilized polymeric micelles (SPMs) with a poly(acrylic acid) (PAA) or a mixed poly(ethylene oxide)/poly(acrylic acid) (PEO/PAA) shell (SPMs with a mixed shell, SPMMS) and a temperature-responsive poly(propylene oxide) (PPO) core was investigated by means of dynamic, static, and electrophoretic light scattering. The SPMs and different types of SPMMS used resulted from the self-assembly of PAA-PPO-PAA triblock copolymer chains, or PAA-PPO-PAA and PEO-PPO-PEO triblock copolymer chain mixtures (with varying chain lengths and molar ratios) in aqueous solutions at pH 10 and the subsequent cross-linking of their PPO cores via loading and photo-cross-linking of pentaerythritol tetraacrylate (PETA). The solution behavior, structure and properties of the formed complexes at pH 7 and 0.01 M ionic strength, were studied as a function of the protein concentration in the solution (the concentration of the stabilized micelles was kept constant) or equivalently the ratio of the two components. The complexation process and properties of the complexes proved to be dependent on the protein concentration, while of particular interest was the effect of the structure of the shell of the SPMs on the stability/solubility of the complexes. Finally, the fluorescence and mid infrared spectroscopic investigation of the structure of the complexed protein showed that, although a small stretching of the protein molecules occurred in some cases, no protein denaturation takes place upon complexation.

  7. Solution-blown nanofiber mats from fish sarcoplasmic protein

    DEFF Research Database (Denmark)

    Sett, S.; Boutrup Stephansen, Karen; Yarin, A.L.


    that the production rate of solution-blowing was increased 30-fold in relation to electrospinning. Overall, this study reveals FSP as an interesting biopolymeric alternative to synthetic polymers, and the introduction of FSP to nylon 6 provides a composite with controlled properties....

  8. Molecular investigation of the interaction between ionic liquid type gemini surfactant and lysozyme: A spectroscopic and computational approach. (United States)

    Maurya, Jitendra Kumar; Mir, Muzaffar Ul Hassan; Singh, Upendra Kumar; Maurya, Neha; Dohare, Neeraj; Patel, Seema; Ali, Anwar; Patel, Rajan


    Herein, we are reporting the interaction of ionic liquid type gemini surfactant, 1,4-bis(3-dodecylimidazolium-1-yl) butane bromide ([C12-4-C12 im]Br2) with lysozyme by using Steady state fluorescence, UV-visible, Time resolved fluorescence, Fourier transform-infrared (FT-IR) spectroscopy techniques in combination with molecular modeling and docking method. The steady state fluorescence spectra suggested that the fluorescence of lysozyme was quenched by [C12-4-C12 im]Br2 through static quenching mechanism as confirmed by time resolved fluorescence spectroscopy. The binding constant for lysozyme-[C12-4-C12 im]Br2 interaction have been measured by UV-visible spectroscopy and found to be 2.541 × 10(5) M(-1). The FT-IR results show conformational changes in the secondary structure of lysozyme by the addition of [C12-4-C12 im]Br2. Moreover, the molecular docking study suggested that hydrogen bonding and hydrophobic interactions play a key role in the protein-surfactant binding. Additionally, the molecular dynamic simulation results revealed that the lysozyme-[C12-4-C12 im]Br2 complex reaches an equilibrium state at around 3 ns.

  9. Biospecific protein immobilization for rapid analysis of weak protein interactions using self-interaction nanoparticle spectroscopy. (United States)

    Bengali, Aditya N; Tessier, Peter M


    "Reversible" protein interactions govern diverse biological behavior ranging from intracellular transport and toxic protein aggregation to protein crystallization and inactivation of protein therapeutics. Much less is known about weak protein interactions than their stronger counterparts since they are difficult to characterize, especially in a parallel format (in contrast to a sequential format) necessary for high-throughput screening. We have recently introduced a highly efficient approach of characterizing protein self-association, namely self-interaction nanoparticle spectroscopy (SINS; Tessier et al., 2008; J Am Chem Soc 130:3106-3112). This approach exploits the separation-dependent optical properties of gold nanoparticles to detect weak self-interactions between proteins immobilized on nanoparticles. A limitation of our previous work is that differences in the sequence and structure of proteins can lead to significant differences in their affinity to adsorb to nanoparticle surfaces, which complicates analysis of the corresponding protein self-association behavior. In this work we demonstrate a highly specific approach for coating nanoparticles with proteins using biotin-avidin interactions to generate protein-nanoparticle conjugates that report protein self-interactions through changes in their optical properties. Using lysozyme as a model protein that is refractory to characterization by conventional SINS, we demonstrate that surface Plasmon wavelengths for gold-avidin-lysozyme conjugates over a range of solution conditions (i.e., pH and ionic strength) are well correlated with lysozyme osmotic second virial coefficient measurements. Since SINS requires orders of magnitude less protein and time than conventional methods (e.g., static light scattering), we envision this approach will find application in large screens of protein self-association aimed at either preventing (e.g., protein aggregation) or promoting (e.g., protein crystallization) these

  10. Evaluation of extraction solutions for biochemical analyses of the proteins in rice grains. (United States)

    Lang, Gang-hua; Kagiya, Yukari; Ohnishi-Kameyama, Mayumi; Kitta, Kazumi


    The influence of different extraction solutions on the proteins extracted from rice grains was investigated. The largest amounts of salt-soluble proteins were extracted with solutions supplemented with Tris-HCl at pH 8.0. Rice allergens were analyzed by multiplex immunodetection. Except for α-globulin extracted with the solutions at pH 8.0, which showed a low-molecular-weight band besides the main band, no significant solution-dependent difference among the allergens was found. Total proteins were extracted with four kinds of solution. The extraction of the basic subunit of glutelin was found to be SDS-dependent, and more protein was obtained with extraction solutions supplemented with SDS. The contents of α-globulin and α-amylase/trypsin inhibitors were higher in the extracts without SDS than with SDS. We conclude from the present data that, in order to obtain comparable data from rice grain salt-soluble and total protein analyses, differences in the protein extraction efficiency of solutions used should be taken into consideration.

  11. The solution structure of an anti-CRISPR protein


    Maxwell, Karen L.; Garcia, Bianca; Bondy-Denomy, Joseph; Bona, Diane; Hidalgo-Reyes, Yurima; Davidson, Alan R


    Bacterial CRISPR–Cas adaptive immune systems use small guide RNAs to protect against phage infection and invasion by foreign genetic elements. We previously demonstrated that a group of Pseudomonas aeruginosa phages encode anti-CRISPR proteins that inactivate the type I-F and I-E CRISPR–Cas systems using distinct mechanisms. Here, we present the three-dimensional structure of an anti-CRISPR protein and map a functional surface that is critical for its potent inhibitory activity. The interacti...

  12. Edwardsiella tarda Ivy, a lysozyme inhibitor that blocks the lytic effect of lysozyme and facilitates host infection in a manner that is dependent on the conserved cysteine residue. (United States)

    Wang, Chong; Hu, Yong-hua; Sun, Bo-guang; Li, Jun; Sun, Li


    Edwardsiella tarda is a Gram-negative bacterial pathogen with a broad host range that includes fish and humans. In this study, we examined the activity and function of the lysozyme inhibitor Ivy (named IvyEt) identified in the pathogenic E. tarda strain TX01. IvyEt possesses the Ivy signature motif CKPHDC in the form of (82)CQPHNC(87) and contains several highly conserved residues, including a tryptophan (W55). For the purpose of virulence analysis, an isogenic TX01 mutant, TXivy, was created. TXivy bears an in-frame deletion of the ivyEt gene. A live infection study in a turbot (Scophthalmus maximus) model showed that, compared to TX01, TXivy exhibited attenuated overall virulence, reduced tissue dissemination and colonization capacity, an impaired ability to replicate in host macrophages, and decreased resistance against the bactericidal effect of host serum. To facilitate functional analysis, recombinant IvyEt (rIvy) and three mutant proteins, i.e., rIvyW55A, rIvyC82S, and rIvyH85D, which bear Ala, Ser, and Asp substitutions at W55, C82, and H85, respectively, were prepared. In vitro studies showed that rIvy, rIvyW55A, and rIvyH85D were able to block the lytic effect of lysozyme on a Gram-positive bacterium, whereas rIvyC82S could not do so. Likewise, rIvy, but not rIvyC82S, inhibited the serum-facilitated killing effect of lysozyme on E. tarda. In vivo analysis showed that rIvy, but not rIvyC82S, restored the lost pathogenicity of TXivy and enhanced the infectivity of TX01. Together these results indicate that IvyEt is a lysozyme inhibitor and a virulence factor that depends on the conserved C82 for biological activity.

  13. Combined effects of lactoferrin and lysozyme on Streptococcus pneumoniae killing. (United States)

    André, G O; Politano, W R; Mirza, S; Converso, T R; Ferraz, L F C; Leite, L C C; Darrieux, M


    Streptococcus pneumoniae is a common colonizer of the human nasopharynx, which can occasionally spread to sterile sites, causing diseases such as otitis media, sinusitis, pneumonia, meningitis and bacteremia. Human apolactoferrin (ALF) and lysozyme (LZ) are two important components of the mucosal innate immune system, exhibiting lytic effects against a wide range of microorganisms. Since they are found in similar niches of the host, it has been proposed that ALF and LZ could act synergistically in controlling bacterial spread throughout the mucosa. The combination of ALF and LZ has been shown to enhance killing of different pathogens in vitro, with ALF facilitating the latter action of LZ. The aim of the present work was to investigate the combined effects of ALF and LZ on S pneumoniae. Concomitant addition of ALF and LZ had a synergistic killing effect on one of the pneumococci tested. Furthermore, the combination of ALF and ALZ was more bactericidal than lysozyme alone in all pneumococcal strains. Pneumococcal surface protein A (PspA), an important vaccine candidate, partially protects pneumococci from ALF mediated killing, while antibodies against one PspA enhance killing of the homologous strain by ALF. However, the serological variability of this molecule could limit the effect of anti-PspA antibodies on different pneumococci. Therefore, we investigated the ability of anti-PspA antibodies to increase ALF-mediated killing of strains that express different PspAs, and found that antisera to the N-terminal region of PspA were able to increase pneumococcal lysis by ALF, independently of the sequence similarities between the molecule expressed on the bacterial surface and that used to produce the antibodies. LF binding to the pneumococcal surface was confirmed by flow cytometry, and found to be inhibited in presence of anti-PspA antibodies. On a whole, the results suggest a contribution of ALF and LZ to pneumococcal clearance, and confirm PspA's ability to interact

  14. Effect of sulfoxides on the thermal denaturation of hen lysozyme: A calorimetric and Raman study (United States)

    Torreggiani, A.; Di Foggia, M.; Manco, I.; De Maio, A.; Markarian, S. A.; Bonora, S.


    A multidisciplinary study of the thermal denaturation of lysozyme in the presence of three sulfoxides with different length in hydrocarbon chain (DMSO, DESO, and DPSO) was carried out by means of DSC, Raman spectroscopy, and SDS-PAGE techniques. In particular, the Td and Δ H values obtained from the calorimetric measurements showed that lysozyme is partially unfolded by sulfoxides but most of the conformation holds native state. The sulfoxide denaturing ability increases in the order DPSO > DESO > DMSO. Moreover, only DMSO and DESO have a real effect in preventing the heat-induced inactivation of the protein and their maximum heat-protective ability is reached when the DMSO and DESO amount is ⩾25% w/w. The sulfoxide ability to act as effective protective agents against the heat-induced inactivation was confirmed by the protein analysis. The enzymatic activity, as well as the SDS-PAGE analysis, suggested that DESO, having a low hydrophobic character and a great ability to stabilise the three-dimensional water structure, is the most heat-protective sulfoxide. An accurate evaluation of the heat-induced conformational changes of the lysozyme structure before and after sulfoxide addition was obtained by the analysis of the Raman spectra. The addition of DMSO or DESO in low concentration resulted to sensitively decrease the heat-induced structural modifications of the protein.

  15. Enhanced antibacterial activity of lysozyme immobilized on chitin nanowhiskers. (United States)

    Jiang, Suisui; Qin, Yang; Yang, Jie; Li, Man; Xiong, Liu; Sun, Qingjie


    In this paper, the contribution of chitin nanowhiskers (CHNW) to the enzymatic activity and antimicrobial activity of lysozyme adsorbed on CHNW was investigated. An activity assay showed that the lysozyme-CHNW system exhibited significant promotion potency on lysozyme activity, which was approximately 1.5-fold greater than that of free lysozyme. The molecular promotion mechanism of lysozyme immobilized by adsorption onto CHNW was investigated by ultraviolet-visible spectrophotometry, fluorescence spectroscopy, and circular dichroism spectroscopy. The results indicated that changes in the secondary structure of lysozyme adsorption onto CHNW resulted in superior enzymatic activity. Furthermore, the antimicrobial assays indicated that the antimicrobial activity of the lysozyme-CHNW system was greater than that of free lysozyme, whereas its antimicrobial effect on a gram-negative bacterium was better than that on gram-positive bacteria. This research provides a facile and promising approach for increasing the application of chitin-derived and enhancing the antibacterial efficiency on preservation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Action of egg white lysozyme on Clostridium tyrobutyricum. (United States)

    Wasserfall, F; Teuber, M


    A 500-U ml-1 portion of egg white lysozyme was able to kill 99% of 5 X 10(5) resting vegetative cells of Clostridium tyrobutyricum within 24 h of incubation at 25 degrees C. Spores were completely resistant to lysozyme. Proliferating vegetative cells were severely inhibited, although lysozyme-resistant cells developed in growing cultures in the presence of lysozyme. Whereas early stages of spore germination (loss of optical refractility and heat resistance) were not inhibited by lysozyme, the overall outgrowth of spore cells into vegetative cells was delayed by 1 day in the presence of 500 U of lysosyme ml-1. This delay was independent of the lysozyme sensitivity or resistance of the mother culture of the used spores. It is suggested that this inhibition by lysozyme of the outgrowth of spore cells into vegetative cells of the lactate-fermenting C. tyrobutyricum is the basis for the observation that lysozyme can substitute for nitrate in preventing the "late gas" defect of Edam- and Gouda-type cheeses.

  17. Co-option of bacteriophage lysozyme genes by bivalve genomes (United States)

    Wang, Chunyang; Jin, Min; Lan, Jiangfeng; Ye, Ting; Hui, Kaimin; Tan, Jingmin; Wang, Zheng; Wang, Wen; Han, Guan-Zhu


    Eukaryotes have occasionally acquired genetic material through horizontal gene transfer (HGT). However, little is known about the evolutionary and functional significance of such acquisitions. Lysozymes are ubiquitous enzymes that degrade bacterial cell walls. Here, we provide evidence that two subclasses of bivalves (Heterodonta and Palaeoheterodonta) acquired a lysozyme gene via HGT, building on earlier findings. Phylogenetic analyses place the bivalve lysozyme genes within the clade of bacteriophage lysozyme genes, indicating that the bivalves acquired the phage-type lysozyme genes from bacteriophages, either directly or through intermediate hosts. These bivalve lysozyme genes underwent dramatic structural changes after their co-option, including intron gain and fusion with other genes. Moreover, evidence suggests that recurrent gene duplication occurred in the bivalve lysozyme genes. Finally, we show the co-opted lysozymes exhibit a capacity for antibacterial action, potentially augmenting the immune function of related bivalves. This represents an intriguing evolutionary strategy in the eukaryote–microbe arms race, in which the genetic materials of bacteriophages are co-opted by eukaryotes, and then used by eukaryotes to combat bacteria, using a shared weapon against a common enemy. PMID:28100665

  18. Optimized Baxter model of protein solutions: electrostatics versus adhesion

    NARCIS (Netherlands)

    Prinsen, P.; Odijk, T.


    A theory is set up of spherical proteins interacting by screened electrostatics and constant adhesion, in which the effective adhesion parameter is optimized by a variational principle for the free energy. An analytical approach to the second virial coefficient is first outlined by balancing the rep

  19. [Molecular cloning, recombinant expression and characterization of lysozyme from Chinese shrimp Fenneropenaeus chinensis]. (United States)

    Bu, Xingjiang; Du, Xinjun; Zhou, Wenjie; Zhao, Xiaofan; Wang, Jinxing


    Lysozyme hydrolyses bacterial cell walls and acts as a nonspecific innate immunity molecule against the invasion of bacterial pathogens. We cloned the cDNA of lysozyme from Fenneropenaeus chinensis and named Fc-lysozyme (FcLyz in short). The full length of the gene was of 709 bp, and the open reading frame (477 bp) encoded 158 amino acids. The predicted protein had a signal peptide (-1--18 residue) and molecular weight of the mature protein (residue 1-140) was of 16.2 kD. A Lyz 1 domain (residue 1-130) in the lysozyme was found by SMART analysis. The results of semiquantity RT-PCR showed that FcLyz was constitutively expressed in tested tissues in a low level in normal shrimp, and up-regulated in hemocytes, heart, hepatopancreas and gill of bacterial challenged shrimp. The DNA fragment of mature Fc-Lys was subcloned to pET-30a (+) expression vector, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and then induced by isopropylthio-beta-D-galactoside (IPTG). The antibacterial activity of the purified recombinant FcLys was analyzed and minimal inhibitory concentration (MIC) was assayed. The recombinant protein showed high antibacterial activity against some Gram-positive bacteria, and MIC reached 3.43 micromol/L, and relatively low activity against Gram-negative bacteria. All together, the Fc-Lys was regulated by pathogen infection and had antibacterial activity. This suggested that the FcLyz may be one of the important molecules against pathogens in innate immunity of the shrimp.

  20. Examination and Manipulation of Protein Surface Charge in Solution with Electrospray Ionization Mass Spectrometry (United States)

    Gross, Deborah S.; Van Ryswyk, Hal


    Electrospray ionization mass spectrometry (ESI-MS) is a powerful tool for examining the charge of proteins in solution. The charge can be manipulated through choice of solvent and pH. Furthermore, solution-accessible, protonated lysine side chains can be specifically tagged with 18-crown-6 ether to form noncovalent adducts. Chemical derivatization…

  1. Examination and Manipulation of Protein Surface Charge in Solution with Electrospray Ionization Mass Spectrometry (United States)

    Gross, Deborah S.; Van Ryswyk, Hal


    Electrospray ionization mass spectrometry (ESI-MS) is a powerful tool for examining the charge of proteins in solution. The charge can be manipulated through choice of solvent and pH. Furthermore, solution-accessible, protonated lysine side chains can be specifically tagged with 18-crown-6 ether to form noncovalent adducts. Chemical derivatization…

  2. Protein-bound solute removal during extended multipass versus standard hemodialysis

    DEFF Research Database (Denmark)

    Eloot, Sunny; Van Biesen, Wim; Axelsen, Mette


    BACKGROUND: Multipass hemodialysis (MPHD) is a recently described dialysis modality, involving the use of small volumes of dialysate which are repetitively recycled. Dialysis regimes of 8 hours for six days a week using this device result in an increased removal of small water soluble solutes and...... times/week 4 h SHD, but at the expense of a lower total solute removal of highly protein-bound solutes....

  3. Comparison of two codon optimization strategies enhancing recombinant Sus scrofa lysozyme production in Pichia pastoris. (United States)

    Zhu, D; Cai, G; Wu, D; Lu, J


    Lysozyme has played an important role in animal feed additive industry, food additive industry and biological engineering. For improving expression efficiency of recombinant lysozyme from Sus scrofa, two genes respectively designed by the most used codon optimization strategies, "one amino acid one codon" and "codon randomization", were synthesized and expressed in Pichia pastoris X—33. At shaking flask level, Sus scrofa lysozyme (SSL) under two conditions had a highest activity of 153.33±10.41 and 538.33±15.18 U/mL after a 5 days induction of 1% methanol, with secreted protein concentration 80.03±1.94 and 239.60±4.16 mg/L, respectively. Compared with the original SSL gene, the expression of optimized SSL gene by the second strategy showed a 2.6 fold higher level, while the first method had no obvious improvement in production. In total secreted protein, the proportions of recombinant SSL encoded by the original gene, first method optimized gene and the second—strategy optimized one were 75.06±0.25%, 74.56±0.14% and 79.00±0.14%, respectively, with the same molecular weight about 18 kDa, optimum acidity pH 6.0 and optimum temperature 35degC.

  4. Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens. (United States)

    Wu, Hanyu; Cao, Dainan; Liu, Tongxin; Zhao, Jianmin; Hu, Xiaoxiang; Li, Ning


    Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY) in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies.

  5. Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens.

    Directory of Open Access Journals (Sweden)

    Hanyu Wu

    Full Text Available Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies.

  6. Solution scattering studies on a virus capsid protein as a building block for nanoscale assemblies

    NARCIS (Netherlands)

    Comellas Aragones, M.; Comellas-Aragones, Marta; Sikkema, Friso D.; Delaittre, Guillaume; Terry, Ann E.; King, Stephen M.; Visser, Dirk; Heenan, Richard K.; Nolte, Roeland J.M.; Cornelissen, Jeroen Johannes Lambertus Maria; Feiters, Martin C.


    Self-assembled protein cages are versatile building blocks in the construction of biomolecular nanostructures. Because of the defined assembly behaviour the cowpea chlorotic mottle virus (CCMV) protein is often used for such applications. Here we report a detailed solution scattering study of the

  7. Evaluation of back scatter interferometry, a method for detecting protein binding in solution

    DEFF Research Database (Denmark)

    Jepsen, S. T.; Jørgensen, Thomas Martini; Zong, Weiyong;


    Back Scatter Interferometry (BSI) has been proposed to be a highly sensitive and versatile refractive index sensor usable for analytical detection of biomarker and protein interactions in solution. However the existing literature on BSI lacks a physical explanation of why protein interactions...

  8. Exercise increases lactoferrin, but decreases lysozyme in salivary granulocytes. (United States)

    Gillum, Trevor; Kuennen, Matthew; McKenna, Zachary; Castillo, Micaela; Jordan-Patterson, Alex; Bohnert, Caitlin


    Intracellular lactoferrin (Lac) and lysozyme (Lys) content play an important role in regulating inflammation and promoting host protection. While exercise has demonstrated an increase in Lac and Lys concentration in exocrine solutions, little is known regarding intracellular concentration changes in response to exercise. To quantify intracellular Lac and Lys concentration before and after exercise in salivary CD45(+)CD15(+) cells. 11 males (20.3 ± 0.8 years, 57.2 ± 7.6 mL/kg/min V̇O2pk, 11.1 ± 3.9% body fat) ran for 45 min at 75% of VO2pk. 12 mL of stimulated saliva were collected pre and immediately post exercise. Saliva was filtered through a 30-µm filter before analysis of leukocytes (CD45(+)) and granulocytes (CD45(+)CD15(+)) using flow cytometry. Median fluorescent intensity (MFI) of Lac increased from pre (64,268 ± 46,036 MFI) to post (117,134 ± 88,115 MFI) exercise (p exercise (pre: 16,933 ± 8249; post: 11,616 ± 6875) (p exercise. Conversely, the exercise-associated decrease of intracellular Lys content could be the cause of increased Lys in exocrine solutions.

  9. Probe conformational dynamics of proteins in aqueous solutions by terahertz spectroscopy (United States)

    Vinh, Nguyen Q.


    Proteins solvated in their biologically milieu are expected to exhibit strong absorption in the terahertz frequencies, that contain information on their global and sub-global collective vibrational modes (conformational dynamics) and global dynamic correlations among solvent water and proteins. The dynamics play an important role in enzymatic activities of proteins, but obtaining an accurate and quantitative pictures of these activities, however, is challenging due to the strong absorption of water. In response, we have developed the world's highest precision, highest sensitivity terahertz-frequency domain spectrometer and a standard terahertz-time domain system to probe the collective dynamics of proteins in aqueous solutions. Operating over the frequency range from 5 GHz up to 3 THz, our spectrometers provide an unparalleled ability to probe directly such questions as the hydration level, the dynamics of water and hydrated proteins over the 100 fs to 1 ns timescale. Employing an effective medium approximation to describe the complex dielectric response of the solvated proteins in solution we find that proteins are surrounded by a loosely and tightly held layers of water molecules that behave as if they are an integral part of the protein. The number of water molecules in the protein hydration shells varies with proteins, which can tell us the average surface structure of proteins. These measurements shed light on the macromolecular motions of proteins in their biologically relevant environment.

  10. Cooperative hydration effect causes thermal unfolding of proteins and water activity plays a key role in protein stability in solutions. (United States)

    Miyawaki, Osato; Dozen, Michiko; Hirota, Kaede


    The protein unfolding process observed in a narrow temperature range was clearly explained by evaluating the small difference in the enthalpy of hydrogen-bonding between amino acid residues and the hydration of amino acid residue separately. In aqueous solutions, the effect of cosolute on the protein stability is primarily dependent on water activity, aw, the role of which has been long neglected in the literature. The effect of aw on protein stability works as a power law so that a small change in aw is amplified substantially through the cooperative hydration effect. In the present approach, the role of hydrophobic interaction stands behind. This affects protein stability indirectly through the change in solution structure caused by the existence of cosolute.

  11. Membrane-protein binding measured with solution-phase plasmonic nanocube sensors. (United States)

    Wu, Hung-Jen; Henzie, Joel; Lin, Wan-Chen; Rhodes, Christopher; Li, Zhu; Sartorel, Elodie; Thorner, Jeremy; Yang, Peidong; Groves, Jay T


    We describe a solution-phase sensor of lipid-protein binding based on localized surface plasmon resonance (LSPR) of silver nanocubes. When silica-coated nanocubes are mixed in a suspension of lipid vesicles, supported membranes spontaneously assemble on their surfaces. Using a standard laboratory spectrophotometer, we calibrated the LSPR peak shift due to protein binding to the membrane surface and then characterized the lipid-binding specificity of a pleckstrin homology domain protein.

  12. Corneal cell adhesion to contact lens hydrogel materials enhanced via tear film protein deposition.

    Directory of Open Access Journals (Sweden)

    Claire M Elkins

    Full Text Available Tear film protein deposition on contact lens hydrogels has been well characterized from the perspective of bacterial adhesion and viability. However, the effect of protein deposition on lens interactions with the corneal epithelium remains largely unexplored. The current study employs a live cell rheometer to quantify human corneal epithelial cell adhesion to soft contact lenses fouled with the tear film protein lysozyme. PureVision balafilcon A and AirOptix lotrafilcon B lenses were soaked for five days in either phosphate buffered saline (PBS, borate buffered saline (BBS, or Sensitive Eyes Plus Saline Solution (Sensitive Eyes, either pure or in the presence of lysozyme. Treated contact lenses were then contacted to a live monolayer of corneal epithelial cells for two hours, after which the contact lens was sheared laterally. The apparent cell monolayer relaxation modulus was then used to quantify the extent of cell adhesion to the contact lens surface. For both lens types, lysozyme increased corneal cell adhesion to the contact lens, with the apparent cell monolayer relaxation modulus increasing up to an order of magnitude in the presence of protein. The magnitude of this increase depended on the identity of the soaking solution: lenses soaked in borate-buffered solutions (BBS, Sensitive Eyes exhibited a much greater increase in cell attachment upon protein addition than those soaked in PBS. Significantly, all measurements were conducted while subjecting the cells to moderate surface pressures and shear rates, similar to those experienced by corneal cells in vivo.

  13. Coated carbon hemoperfusion provides limited clearance of protein-bound solutes. (United States)

    Dinh, Diana C; Recht, Natalie S; Hostetter, Thomas H; Meyer, Timothy W


    This study assessed the capacity of a cartridge containing coated granular carbon to clear protein-bound solutes. Clearances for test solutes were measured while an albumin solution representing plasma was pumped from a 10 L reservoir through the cartridge at a rate of 200 mL/min for 5 h. Clearance values for phenol red, phenytoin, and indican were well below the limit imposed by the plasma flow and declined with time. The clearance of phenol red, which was the most tightly bound solute, fell from 38 +/- 12 to 17 +/- 2 mL/min. Additional studies revealed that the cartridge contained enough carbon to absorb all the protein-bound test solutes, but that the rate of their clearance was limited by the inability of granular carbon to take up solutes rapidly at a low concentration. The rate of solute uptake at low concentration was shown to be much greater when carbon was in powdered rather than granular form. A device in which approximately 50 g of powdered carbon was recirculated in the dialysate compartment of hollow fiber kidneys cleared phenol red and phenytoin more rapidly than the hemoperfusion cartridge containing 300 g of coated granular carbon. These results indicate that hemoperfusion over coated granular carbon provides limited clearance of protein-bound solutes.

  14. [Purification and characterization of a lysozyme from a marine microorganism]. (United States)

    Zou, Yan-Li; Sun, Mi; Wang, Yue-Jun


    A novel lysozyme was purified from a marine microorganism and its major characteristics were studied. Cell-free supernatant was prepared by centrifugation of culture broth, ultrafiltration using a hollow fiber (molecular weight cut off, 50kD) and concentration using a hollow fiber (molecular weight cut off, 10kD). The crude lysozyme was purified 34.7 fold to electrophoretic homogeneity with a recovery of 24.1% by CM-Sepharose FF cationic-exchange and Sephadex G-100 gel chromatography. The relative molecular weight of this lysozyme was determined as about 39 kD. The optimum pH and temperature towards Micrococcus lysodleikticus were pH 8.0 and 35 degrees C respectively, and the enzyme was stable at temperature below 50 degrees C and pH 5.0 - 10.0. The lysozyme activity was slightly enhanced by Zn2+ and Cu2+ and slightly inhibited by Mn2+ and Ag+. The lysozyme showed good compatibility to many common chemical agents such as EDTA (0.1%) and KH2 PO4 (1.0%). The lysozyme had broad-spectrum against many bacteria, including a number of pathogens, which were resistant to egg-white lysozyme.

  15. Thermal contraction of aqueous glycerol and ethylene glycol solutions for optimized protein-crystal cryoprotection. (United States)

    Shen, Chen; Julius, Ethan F; Tyree, Timothy J; Moreau, David W; Atakisi, Hakan; Thorne, Robert E


    The thermal contraction of aqueous cryoprotectant solutions on cooling to cryogenic temperatures is of practical importance in protein cryocrystallography and in biological cryopreservation. In the former case, differential contraction on cooling of protein molecules and their lattice relative to that of the internal and surrounding solvent may lead to crystal damage and the degradation of crystal diffraction properties. Here, the amorphous phase densities of aqueous solutions of glycerol and ethylene glycol at T = 77 K have been determined. Densities with accuracies of solutions. The use of these densities in contraction matching of internal solvent to the available solvent spaces is complicated by several factors, most notably the exclusion of cryoprotectants from protein hydration shells and the expected deviation of the contraction behavior of hydration water from bulk water. The present methods and results will assist in developing rational approaches to cryoprotection and an understanding of solvent behavior in protein crystals.

  16. Development of SH-SAW sensors for measurement of the properties of protein solutions (United States)

    Roh, Yongrae; Kwon, Yongjun; Kim, Kyungho; Koh, Kwangnak; Kim, Jaeho


    We developed new surface acoustic wave (SAW) sensors to measure the properties of protein solutions applying a particular organic thin film on the delay line of transverse type SAW devices. Each delay line is configured as an oscillator. The delay line for a sensing channel is coated with a gold film on which an antibody layer is immobilized by protein A. The sensing delay line selectively adsorbs antigens when exposed to a protein solution, which results in phase delay changes due to the mass loading effects induced by the adsorbed antigens. The other delay line is uncoated for use as a stable reference. The relative change in the frequency of the two oscillators is monitored to measure the concentration of the antigens. Sensor properties investigated include selectivity, sensitivity, response time and stability in response to the antigen concentration as well as the viscosity and electrical conductivity of the protein solution.

  17. Solution Properties of Murine Leukemia Virus Gag Protein: Differences from HIV-1 Gag

    Energy Technology Data Exchange (ETDEWEB)

    Datta, Siddhartha A.K.; Zuo, Xiaobing; Clark, Patrick K.; Campbell, Stephen J.; Wang, Yun-Xing; Rein, Alan (SAIC); (NCI)


    Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV), a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of {approx}7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed.

  18. Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries. (United States)

    Pröpper, Kevin; Meindl, Kathrin; Sammito, Massimo; Dittrich, Birger; Sheldrick, George M; Pohl, Ehmke; Usón, Isabel


    Protein-DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein-DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite the fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein-DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein-DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.

  19. Arrest scenarios in concentrated protein solutions - from hard sphere glasses to arrested spinodal decomposition (United States)

    Stradner, Anna; Bucciarelli, Saskia; Casal, Lucia; Foffi, Giuseppe; Thurston, George; Farago, Bela; Schurtenberger, Peter


    The occurrence of an arrest transition in concentrated colloid suspensions and its dependence on the interaction potential is a hot topic in soft matter. Such arrest transitions can also occur in concentrated protein solutions, as they exist e.g. in biological cells or are increasingly used in pharmaceutical formulations. Here we demonstrate the applicability of concepts from colloid science to understand the dynamics of concentrated protein solutions. In this presentation we report a combination of 3D light scattering, small-angle X-ray scattering and neutron spin echo measurements to study the structural properties as well as the collective and self diffusion of proteins in highly concentrated solutions on the relevant length and time scales. We demonstrate that various arrest scenarios indeed exist for different globular proteins. The proteins chosen are different bovine lens crystallins. We report examples of hard and attractive glass transitions and arrested spinodal decomposition directly linked to the effective pair potentials determined in static scattering experiments for the different proteins. We discuss these different arrest scenarios in view of possible applications of dense protein solutions as well as in view of their possible relevance for living systems.

  20. Protein structural dynamics revealed by time-resolved X-ray solution scattering. (United States)

    Kim, Jong Goo; Kim, Tae Wu; Kim, Jeongho; Ihee, Hyotcherl


    One of the most important questions in biological science is how a protein functions. When a protein performs its function, it undergoes regulated structural transitions. In this regard, to better understand the underlying principle of a protein function, it is desirable to monitor the dynamic evolution of the protein structure in real time. To probe fast and subtle motions of a protein in physiological conditions demands an experimental tool that is not only equipped with superb spatiotemporal resolution but also applicable to samples in solution phase. Time-resolved X-ray solution scattering (TRXSS), discussed in this Account, fits all of those requirements needed for probing the movements of proteins in aqueous solution. The technique utilizes a pump-probe scheme employing an optical pump pulse to initiate photoreactions of proteins and an X-ray probe pulse to monitor ensuing structural changes. The technical advances in ultrafast lasers and X-ray sources allow us to achieve superb temporal resolution down to femtoseconds. Because X-rays scatter off all atomic pairs in a protein, an X-ray scattering pattern provides information on the global structure of the protein with subangstrom spatial resolution. Importantly, TRXSS is readily applicable to aqueous solution samples of proteins with the aid of theoretical models and therefore is well suited for investigating structural dynamics of protein transitions in physiological conditions. In this Account, we demonstrate that TRXSS can be used to probe real-time structural dynamics of proteins in solution ranging from subtle helix movement to global conformational change. Specifically, we discuss the photoreactions of photoactive yellow protein (PYP) and homodimeric hemoglobin (HbI). For PYP, we revealed the kinetics of structural transitions among four transient intermediates comprising a photocycle and, by applying structural analysis based on ab initio shape reconstruction, showed that the signaling of PYP involves

  1. Linear sweep voltammetric studies on the supramolecular complex of alizarin red S with lysozyme and determination of lysozyme

    Indian Academy of Sciences (India)

    Wei Sun; Na Zhao; Xueliang Niu; Yan Wang; Kui Jiao


    An electrochemical method for the determination of lysozyme (LYS) based on its interaction with alizarin red S (ARS) was established by linear sweep voltammetry in this paper. The electrochemical behaviour of ARS with LYS was investigated on a dropping mercury working electrode in 0.2 mol/L pH 4.8 Britton-Robinson (B-R) buffer solution. ARS showed a sensitive second order derivative linear sweep voltammetric reductive peak at -0.42 V (vs SCE). After the addition of LYS, the reductive peak current of ARS decreased without the shift of the reductive peak potential and no new waves appeared, which was due to the formation of a supramolecular complex of ARS with LYS in the solution. The stoichiometry of the ARS-LYS complex was further calculated by the electrochemical data with the results of the binding ratio as 3 : 1 and the binding constant as 2.82 × 1014. Under the selected conditions, the decrease of the second order derivative linear sweep voltammetric reductive peak current of ARS was in proportion to the LYS concentration in the range from 0.8 to 35.0 mg/L and the detection limit of LYS was calculated as 0.52 mg/L (3). Different kinds of LYS samples were detected satisfactorily with this method.

  2. Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode. (United States)

    To, Brian C S; Lenhoff, Abraham M


    The adsorption isotherms of four model proteins (lysozyme, α-lactalbumin, ovalbumin, and BSA) on eight commercial phenyl hydrophobic interaction chromatography media were measured. The isotherms were softer than those usually seen in ion-exchange chromatography of proteins, and the static capacities of the media were lower, ranging from 30 to 110 mg/mL, depending on the ammonium sulfate concentration and the protein and adsorbent types. The protein-accessible surface area appears to be the main factor determining the binding capacity, and little correlation was seen with the protein affinities of the adsorbents. Breakthrough experiments showed that the dynamic capacities of the adsorbents at 10% breakthrough were 20-80% of the static capacities, depending on adsorbent type. Protein diffusivities in the adsorbents were estimated from batch uptake experiments using the pore diffusion and homogeneous diffusion models. Protein transport was affected by the adsorbent pore structures. Apparent diffusivities were higher at lower salt concentrations and column loadings, suggesting that adsorbed proteins may retard intraparticle protein transport. The diffusivities estimated from the batch uptake experiments were used to predict column breakthrough behavior. Analytical solutions developed for ion-exchange systems were able to provide accurate predictions for lysozyme breakthrough but not for ovalbumin. Impurities in the ovalbumin solutions used for the breakthrough experiments may have affected the ovalbumin uptake and led to the discrepancies between the predictions and the experimental results.

  3. Molecular dynamics investigation of desorption and ion separation following picosecond infrared laser (PIRL) ablation of an ionic aqueous protein solution (United States)

    Zou, J.; Wu, C.; Robertson, W. D.; Zhigilei, L. V.; Miller, R. J. D.


    Molecular dynamics simulations were performed to characterize the ablation process induced by a picosecond infrared laser (PIRL) operating in the regime of desorption by impulsive vibrational excitation (DIVE) of a model peptide (lysozyme)/counter-ion system in aqueous solution. The simulations were performed for ablation under typical experimental conditions found within a time-of-flight mass spectrometer (TOF-MS), that is in vacuum with an applied electric field (E = ± 107 V/m), for up to 2 ns post-ablation and compared to the standard PIRL-DIVE ablation condition (E = 0 V/m). Further, a simulation of ablation under an extreme field condition (E = 1010 V/m) was performed for comparison to extend the effective dynamic range of the effect of the field on charge separation. The results show that the plume dynamics were retained under a typical TOF-MS condition within the first 1 ns of ablation. Efficient desorption was observed with more than 90% of water molecules interacting with lysozyme stripped off within 1 ns post-ablation. The processes of ablation and desolvation of analytes were shown to be independent of the applied electric field and thus decoupled from the ion separation process. Unlike under the extreme field conditions, the electric field inside a typical TOF-MS was shown to modify the ions' motion over a longer time and in a soft manner with no enhancement to fragmentation observed as compared to the standard PIRL-DIVE. The study indicates that the PIRL-DIVE ablation mechanism could be used as a new, intrinsically versatile, and highly sensitive ion source for quantitative mass spectrometry.

  4. Molecular dynamics investigation of desorption and ion separation following picosecond infrared laser (PIRL) ablation of an ionic aqueous protein solution. (United States)

    Zou, J; Wu, C; Robertson, W D; Zhigilei, L V; Miller, R J D


    Molecular dynamics simulations were performed to characterize the ablation process induced by a picosecond infrared laser (PIRL) operating in the regime of desorption by impulsive vibrational excitation (DIVE) of a model peptide (lysozyme)/counter-ion system in aqueous solution. The simulations were performed for ablation under typical experimental conditions found within a time-of-flight mass spectrometer (TOF-MS), that is in vacuum with an applied electric field (E = ± 10(7) V/m), for up to 2 ns post-ablation and compared to the standard PIRL-DIVE ablation condition (E = 0 V/m). Further, a simulation of ablation under an extreme field condition (E = 10(10) V/m) was performed for comparison to extend the effective dynamic range of the effect of the field on charge separation. The results show that the plume dynamics were retained under a typical TOF-MS condition within the first 1 ns of ablation. Efficient desorption was observed with more than 90% of water molecules interacting with lysozyme stripped off within 1 ns post-ablation. The processes of ablation and desolvation of analytes were shown to be independent of the applied electric field and thus decoupled from the ion separation process. Unlike under the extreme field conditions, the electric field inside a typical TOF-MS was shown to modify the ions' motion over a longer time and in a soft manner with no enhancement to fragmentation observed as compared to the standard PIRL-DIVE. The study indicates that the PIRL-DIVE ablation mechanism could be used as a new, intrinsically versatile, and highly sensitive ion source for quantitative mass spectrometry.

  5. [Generation of transgenic mice expressing human lysozyme in mammary gland]. (United States)

    Yan, Hua; Li, Guo-cai; Sun, Huai-chang


    To evaluate the feasibility of generating animal mammary gland bioreactors expressing human lysozyme (hLYZ). The recombinant vector p205C3-hLYZ, as a result of connecting the hLYZ cDNA with the mammry gland expression vector p205C3, was used to generate transfer genic mice by microinjection. A total of 136 F0 mice were obtained, of which 7 (2 females and 5 males) and 4 (1 females and 3 males) were found to contain the transfer-gene by PCR and Southern blotting respectively. The results of Western blotting indicated that the expressed protein had the same molecular weight as that of normal hLYZ. From the F1 generation on, the mice mated only with their brothers or sisters and a colony of F7 transgenic mice was obtained. Among the offspring, the female transgenic mice maintained and expressed the transfer-gene stably with an expression level as high as 750 mg/L. The expressed protein had strong tissue specificity, and in addition to the mammary glands, some degree of ectropic expression in the spleens and intestines of the transgenic mice was confirmed by dot blotting assay. These data indicate that the mice mammary gland bioreactors expressing hLYZ have been successfully generated.

  6. Measurements of thermal conductivity and thermal diffusivity of hen egg-white lysozyme crystals using a short hot wire method (United States)

    Fujiwara, Seiji; Maki, Syou; Tanaka, Seiichi; Maekawa, Ryunosuke; Masuda, Tomoki; Hagiwara, Masayuki


    Thermal conductivity and thermal diffusivity of hen egg-white lysozyme (HEWL) crystals were examined by using the transient short hot wire method. This method is based on the conventional hot wire method, but improved by using a wire that is much shorter than conventional ones. The magneto-Archimedes levitation technique was utilized to attach the HEWL crystals onto the wire. Owing to the upward magnetic force, the HEWL crystals were deposited at the air-liquid interface of the protein buffer solution where the short hot wire was preliminarily fixed. In situ observation clarified that the wire was completely buried into the HEWL crystals. By means of these techniques, the measurement of thermal conductivity and thermal diffusivity of HEWL crystals was realized for the first time. Gadolinium chloride (a paramagnetic subject) was used as a precipitant agent of crystallization. Crystal growth was carried out over 20 h at 17.2 °C. The applied magnetic field was 4 T. Measurements were conducted during the crystal growth at two different times. The thermal conductivity and diffusivity of the HEWL crystals were determined to be 0.410 W/(m.K) and 3.77×10-8 m2/s at 14 h after, and 0.438 W/(m.K) and 5.18×10-8 m2/s at 20 h after, respectively. We emphasize that this method is versatile and applicable for other protein crystals.

  7. A strategy for selecting the pH of protein solutions to enhance crystallization. (United States)

    Zhang, Chen Yan; Wu, Zi Qing; Yin, Da Chuan; Zhou, Bo Ru; Guo, Yun Zhu; Lu, Hui Meng; Zhou, Ren Bin; Shang, Peng


    The pH of a solution is an important parameter in crystallization that needs to be controlled in order to ensure success. The actual pH of the crystallization droplet is determined by the combined contribution of the buffers in the screening and protein solutions, although the contribution of the latter to the pH is often ignored. In this study, the effects of the buffer and protein solution pH values on the results of screening are systematically investigated. It was found that these parameters significantly affected the results and thus the following strategy for the selection of appropriate pH values is proposed: (i) when screening with only one protein solution, the pH should be as low, as high or as divergent from the pI as possible for a basic, acidic or neutral protein, respectively, within its stable pH range; (ii) when screening with two protein solutions, the pH values should be well separated from one another; and (iii) when multiple pH values are utilized, an even distribution of pH values is the best approach to increase the success rate of crystallization.

  8. Rational design of solution additives for the prevention of protein aggregation. (United States)

    Baynes, Brian M; Trout, Bernhardt L


    We have developed a statistical-mechanical model of the effect of solution additives on protein association reactions. This model incorporates solvent radial distribution functions obtained from all-atom molecular dynamics simulations of particular proteins into simple models of protein interactions. In this way, the effects of additives can be computed along the entire association/dissociation reaction coordinate. We used the model to test our hypothesis that a class of large solution additives, which we term "neutral crowders," can slow protein association and dissociation by being preferentially excluded from protein-protein encounter complexes, in a manner analogous to osmotic stress. The magnitude of this proposed "gap effect" was probed for two simple model systems: the association of two spheres and the association of two planes. Our results suggest that for a protein of 20 A radius, an 8 A additive can increase the free energy barrier for association and dissociation by as much as 3-6 kcal/mol. Because the proposed gap effect is present only for reactions involving multiple molecules, it can be exploited to develop novel additives that affect protein association reactions although having little or no effect on unimolecular reactions such as protein folding. This idea has many potential applications in areas such as the stabilization of proteins against aggregation during folding and in pharmaceutical formulations.

  9. Parallel β-sheet vibration band increases with proteins dipole moment under exposure to 1765 MHz microwaves. (United States)

    Calabrò, Emanuele; Magazù, Salvatore


    Effects of exposure of 4 h to mobile phones microwaves at 1765 MHz at a power density around 940 mW/m(2) on four typical proteins (hemoglobin in H2 O solution, and myoglobin, bovine serum albumin, and lysozyme in D2 O solution) were studied by means of Fourier Transform Infrared spectroscopy and Fourier self-deconvolution analysis. Increase in intensity of parallel β-sheet component around 1635 cm(-1) was observed after exposure of hemoglobin, myoglobin, and bovine serum albumin, showing that a mechanism of unfolding occurred after exposure, whereas no appreciable change in the amide I region occurred after lysozyme exposure. In addition, a relationship between protein dipole moment and protein unfolding rate was demonstrated with a correlation coefficient r = 0.973 and 95% confidence interval.

  10. Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions. (United States)

    Cao, Enhong; Chen, Yahuei; Cui, Zhanfeng; Foster, Peter R


    The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1 degrees C/min) and fast thawing (at a thawing rate >10 degrees C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20 degrees C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol.

  11. Interactions between high pressure homogenization and antimicrobial activity of lysozyme and lactoperoxidase. (United States)

    Vannini, L; Lanciotti, R; Baldi, D; Guerzoni, M E


    It was the objective of this work to evaluate the effect of high pressure homogenization on the activity of antimicrobial enzymes such as lysozyme and lactoperoxidase against a selected group of Gram positive and Gram negative species inoculated in skim milk. Lactobacillus helveticus, Lactobacillus plantarum and Listeria monocytogenes were the most pressure resistant species while Bacillus subtilis, Pseudomonas putida, Salmonella typhimurium, Staphylococcus aureus, Proteus vulgaris and Salmonella enteritidis were found to be very sensitive to the hyperbaric treatment. The enzyme addition enhanced the instantaneous pressure efficacy on almost all the considered species as indicated by their instantaneous viability loss following the treatment. Moreover, the combination of the enzyme and high pressure homogenization significantly affected the recovery and growth dynamics of several of the considered species. Although L. monocytogenes was slightly sensitive to pressure, the combination of the two stress factors induced a significant viability loss within 3 h and an extension of lag phases in skim milk during incubation at 37 degrees C. The hypothesis formulated in this work is that the interaction of high pressure homogenization and lysozyme or lactoperoxidase is associated to conformational modifications of the two proteins with a consequent enhancement of their activity. This hypothesis is supported by the experimental results also regarding the increased antimicrobial activity against L. plantarum of the previously pressurised lysozyme with respect to that of the native enzyme.

  12. Structural relationships in the lysozyme superfamily: significant evidence for glycoside hydrolase signature motifs.

    Directory of Open Access Journals (Sweden)

    Alexandre Wohlkönig

    Full Text Available BACKGROUND: Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily. RESULTS: Besides having a similar fold, all known catalytic domains of glycoside hydrolase proteins of lysozyme superfamily (families and subfamilies GH19, GH22, GH23, GH24 and GH46 share in common two structural elements: the central helix of the all-α domain, which invariably contains the catalytic glutamate residue acting as general-acid catalyst, and a β-hairpin pointed towards the substrate binding cleft. The invariant β-hairpin structure is interestingly found to display the highest amino acid conservation in aligned sequences of a given family, thereby allowing to define signature motifs for each GH family. Most of such signature motifs are found to have promising performances for searching sequence databases. Our structural analysis further indicates that the GH motifs participate in enzymatic catalysis essentially by containing the catalytic water positioning residue of inverting mechanism. CONCLUSIONS: The seven families and subfamilies of the lysozyme superfamily all have in common a β-hairpin structure which displays a family-specific sequence motif. These GH β-hairpin motifs contain potentially important residues for the catalytic activity, thereby suggesting the participation of the GH motif to catalysis and also revealing a common catalytic scheme utilized by enzymes of the lysozyme superfamily.


    Institute of Scientific and Technical Information of China (English)

    SUN Yishi; QIU Zhiyong; HONG Yaoliang


    Six different N-alkyl substituted acrylamide nonionic hydrogels were prepared and their swelling characteristics were measured. Poly N-isopropyl acrylamide (PNIPA) and poly N-n-propylacrylamide (PNNPA) temperature sensitive hydrogels were chosen as the nonionic temperature sensitive hydrogels for concentration of very dilute aqueous protein solution. The separation properties of PNIPA and PNNPA hydrogels with different network dimensions were studied and the modification of the hydrogels was surveyed in order to decrease their surface adsorption of protein molecules. The experimental results of the concentration of BSA (Bovin serum albumin) dilute aqueous solution by hydroxylpropyl methacrylate (HPMA) copolymerized PNIPA hydrogel were given. The value and the limitation of concentration of dilute aqueous protein solution by this method was evaluated.

  14. Antimicrobial activity of lysozyme with special relevance to milk

    African Journals Online (AJOL)



    Dec 29, 2008 ... Lysozyme is a hydrolytic enzyme which has been purified from cells, secretions and tissues of virtually ... breakdown of peptidoglycan polymers of bacterial cell ...... Small Rum. ..... myeloid leukemia: An analysis of 208 cases.

  15. Bioengineered lysozyme in combination therapies for Pseudomonas aeruginosa lung infections (United States)

    Griswold, Karl E; Bement, Jenna L; Teneback, Charlotte C; Scanlon, Thomas C; Wargo, Matthew J; Leclair, Laurie W


    There is increasing urgency in the battle against drug-resistant bacterial pathogens, and this public health crisis has created a desperate need for novel antimicrobial agents. Recombinant human lysozyme represents one interesting candidate for treating pulmonary infections, but the wild type enzyme is subject to electrostatic mediated inhibition by anionic biopolymers that accumulate in the infected lung. We have redesigned lysozyme’s electrostatic potential field, creating a genetically engineered variant that is less susceptible to polyanion inhibition, yet retains potent bactericidal activity. A recent publication demonstrated that the engineered enzyme outperforms wild type lysozyme in a murine model of Pseudomonas aeruginosa lung infection. Here, we expand upon our initial studies and consider dual therapies that combine lysozymes with an antimicrobial peptide. Consistent with our earlier results, the charge modified lysozyme combination outperformed its wild type counterpart, yielding more than an order-of-magnitude reduction in bacterial burden following treatment with a single dose. PMID:24637705

  16. Use of lysozyme from chicken egg white as a nitrite replacer in an Italian-type chicken sausage

    Directory of Open Access Journals (Sweden)

    Nalaka Sandun Abeyrathne


    Full Text Available Background: Sodium or potassium nitrite is widely used as a curing agent in sausages and other cured meat products. Nitrite has strong antimicrobial and antioxidant effects and generates cured meat color. Nitrite, however, can react with secondary or tertiary amines in meat to form carcinogenic, teratogenic and mutagenic N-nitroso compounds. Several findings have been suggested that high consumption of processed meat may increase the risk of cancer, and emphasized that dietary nitrosamines are positively associated with cancer. Lysozyme is one of the major egg proteins that have antimicrobial and antioxidant characteristics. Therefore, lysozyme can be used in meat processing to prevent microbial growth and oxidative degradation in meat products during storage. This study is focused on evaluating the antimicrobial and antioxidant effects of lysozyme extracted from egg white as a replacer of nitrite in a cooked Italian-type chicken sausage. Methods: Four curing treatments including 100% nitrite (control, 100% lysozyme (treatment 1, 25% nitrite + 75% lysozyme (treatment 2 and 50% nitrite + 50% lysozyme (treatment 3 were used to prepare Italian-type chicken sausage samples. Recipe was developed with 64% (w/w meat, 17% (w/w binder (bread crumble, 12% (w/w ice, 4% (w/w vegetable oil, 2% (w/w salt, 1% (w/w spices (chili, black pepper, cardamom. Prepared samples were cooked in an 80 °C smoke house to a core temperature of 65 °C and cooled in cold water to 20-25 °C subsequently packed in polyethylene and stored in a freezer (-18 °C. The antimicrobial effect lysozyme was tested using Escherichia coli and Salmonella. The growth of these pathogens at 0, 3 and 5 days of storage of spore inoculation was determined. The antioxidant activity of lysozyme was determined using the TBARS value during the 25 d storage period. The redness (a*, lightness (L*, and yellowness (b* of sausages were analyzed using a Minolta color meter (CR 410, Konica Minolta Inc

  17. Direct Biomolecules Binding on Nonfouling Surface via Newly Discovered Supramolecular Self-assembly of Lysozyme under Physiological Condition (United States)

    Yang, Peng


    A major challenge in the development of low cost and practical strategies for biomolecules immobilization on solid supports is that the multi-step chemical/physical activating and following deactivating procedures on nonfouling substrates often increase the cost and complexity of surface functional group types as well as deteriorate the surface integrity. Herein, we show a novel phase transition of lysozyme could be used to constitute a major step to address the above problem. It is found that when lysozyme is dissolved in a neutral buffer solution of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 7.4) with 1–50 mM tris(2-carboxyethyl)phosphine (TCEP) added, a fast phase transition process occurs and the resulting novel fibra-like hierarchical supramolecular assemblies made by primary spherical particles aggregation would function as a “superglue” that strongly and quickly bind onto non-fouling coatings. This binding is highly selective towards lysozyme, and excludes completely tedious synthetical, chemical/physical activation/deactivation (blocking) steps. When biotin is conjugated with lysozyme, such phase transition quickly constructs a perfect biotinylated surface on nonfouling surface for avidin binding, showing great potential for the development of low-cost and practical biochips. PMID:22707360

  18. Slowdown of water diffusion around protein in aqueous solution with ectoine (United States)

    Yu, I.; Nagaoka, M.


    Ectoine is one of the most common compatible solutes found in halophilic bacteria, and has an effect to introduce a tolerance to high salt concentration or high temperature. By analyzing 1 ns molecular dynamics simulations at 370 K, we have shown that, in the ectoine aqueous solution, the water diffusion slows down around a protein (chymotrypsin inhibitor 2 (CI2)), keeping the protein hydration structure essentially unchanged. It is concluded that the slowdown of water diffusion around the backbone amide protons must be one of the decisive factors in reducing the exchange rate of the backbone amide protons, whose reduction is experimentally believed closely related to the tolerance effect.

  19. Magnetic Resonance Water Proton Relaxation in Protein Solutions and Tissue: T1ρ Dispersion Characterization (United States)

    Chen, Enn-Ling; Kim, Raymond J.


    Background Image contrast in clinical MRI is often determined by differences in tissue water proton relaxation behavior. However, many aspects of water proton relaxation in complex biological media, such as protein solutions and tissue are not well understood, perhaps due to the limited empirical data. Principal Findings Water proton T1, T2, and T1ρ of protein solutions and tissue were measured systematically under multiple conditions. Crosslinking or aggregation of protein decreased T2 and T1ρ, but did not change high-field T1. T1ρ dispersion profiles were similar for crosslinked protein solutions, myocardial tissue, and cartilage, and exhibited power law behavior with T1ρ(0) values that closely approximated T2. The T1ρ dispersion of mobile protein solutions was flat above 5 kHz, but showed a steep curve below 5 kHz that was sensitive to changes in pH. The T1ρ dispersion of crosslinked BSA and cartilage in DMSO solvent closely resembled that of water solvent above 5 kHz but showed decreased dispersion below 5 kHz. Conclusions Proton exchange is a minor pathway for tissue T1 and T1ρ relaxation above 5 kHz. Potential models for relaxation are discussed, however the same molecular mechanism appears to be responsible across 5 decades of frequencies from T1ρ to T1. PMID:20052404

  20. Constrained solution scattering modelling of human antibodies and complement proteins reveals novel biological insights. (United States)

    Perkins, Stephen J; Okemefuna, Azubuike I; Nan, Ruodan; Li, Keying; Bonner, Alexandra


    X-ray and neutron-scattering techniques characterize proteins in solution and complement high-resolution structural studies. They are useful when either a large protein cannot be crystallized, in which case scattering yields a solution structure, or a crystal structure has been determined and requires validation in solution. These solution structures are determined by the application of constrained modelling methods based on known subunit structures. First, an appropriate starting model is generated. Next, its conformation is randomized to generate thousands of models for trial-and-error fits. Comparison with the experimental data identifies a small family of best-fit models. Finally, their significance for biological function is assessed. We illustrate this in application to structure determinations for secretory immunoglobulin A, the most prevalent antibody in the human body and a first line of defence in mucosal immunity. We also discuss the applications to the large multi-domain proteins of the complement system, most notably its major regulator factor H, which is important in age-related macular degeneration and renal diseases. We discuss the importance of complementary data from analytical ultracentrifugation, and structural studies of protein-protein complexes. We conclude that constrained scattering modelling makes useful contributions to our understanding of antibody and complement structure and function.

  1. Stealth carriers for low-resolution structure determination of membrane proteins in solution

    DEFF Research Database (Denmark)

    Maric, Selma; Skar-Gislinge, Nicholas; Midtgaard, Søren;


    Structural studies of membrane proteins remain a great experimental challenge. Functional reconstitution into artificial nanoscale bilayer disc carriers that mimic the native bilayer environment allows the handling of membrane proteins in solution. This enables the use of small-angle scattering...... techniques for fast and reliable structural analysis. The difficulty with this approach is that the carrier discs contribute to the measured scattering intensity in a highly nontrivial fashion, making subsequent data analysis challenging. Here, an elegant solution to circumvent the intrinsic complexity......O at the length scales relevant to SANS. These 'stealth' carrier discs may be used as a general platform for low-resolution structural studies of membrane proteins using well established data-analysis tools originally developed for soluble proteins. © 2014 International Union of Crystallography....

  2. Development of a stealth carrier system for structural studies of membrane proteins in solution

    DEFF Research Database (Denmark)

    Maric, Selma

    which can be used for SANS structural analysis of membrane proteins in solution. In combination with the D2O/H2O-based contrast variation method it is demonstrated that it is possible to prepare specifically deuterated analogues of the nanodisc, which give minimal contribution to the neutron scattering......Structural studies of membrane proteins remain a great experimental challenge. Functional reconstitution into artificial carriers that mimic the native bilayer environment allows for the handling of membrane proteins in solution and enables the use of small-angle scattering techniques for fast...... scaffolding protein. To obtain physiologically relevant deuterated phosphatidylcholine (PC) species with the required scattering length density a novel method for deuteration of PC was developed to separately control the deuteration levels of three different parts of the phospholipid molecule: the lipid head...

  3. Ancestral Protein Reconstruction Yields Insights into Adaptive Evolution of Binding Specificity in Solute-Binding Proteins. (United States)

    Clifton, Ben E; Jackson, Colin J


    The promiscuous functions of proteins are an important reservoir of functional novelty in protein evolution, but the molecular basis for binding promiscuity remains elusive. We used ancestral protein reconstruction to experimentally characterize evolutionary intermediates in the functional expansion of the polar amino acid-binding protein family, which has evolved to bind a variety of amino acids with high affinity and specificity. High-resolution crystal structures of an ancestral arginine-binding protein in complex with l-arginine and l-glutamine show that the promiscuous binding of l-glutamine is enabled by multi-scale conformational plasticity, water-mediated interactions, and selection of an alternative conformational substate productive for l-glutamine binding. Evolution of specialized glutamine-binding proteins from this ancestral protein was achieved by displacement of water molecules from the protein-ligand interface, reducing the entropic penalty associated with the promiscuous interaction. These results provide a structural and thermodynamic basis for the co-option of a promiscuous interaction in the evolution of binding specificity.

  4. A direct comparison of protein structure in the gas and solution phase: the Trp-cage

    DEFF Research Database (Denmark)

    Patriksson, Alexandra; Adams, Christopher M; Kjeldsen, Frank;


    Molecular dynamics simulations of zwitterions of the Trp-cage protein in the gas phase show that the most stable ion in vacuo has preserved the charge locations acquired in solution. A direct comparison of the gas and solution-phase structures reveals that, despite the similarity in charge location......, there is significant difference in the structures, with a substantial increase in hydrogen bonds and exposure of hydrophobic parts in the gas phase. The structure of the salt bridge in the gas phase is also much more stable than in the (experimental) solution structure....

  5. Assessment of the Protein-Protein Interactions in a Highly Concentrated Antibody Solution by Using Raman Spectroscopy. (United States)

    Ota, Chikashi; Noguchi, Shintaro; Nagatoishi, Satoru; Tsumoto, Kouhei


    To investigate the protein-protein interactions of a highly concentrated antibody solution that could cause oligomerization or aggregation and to develop a better understanding of the optimization of drug formulations. In this study, we used Raman spectroscopy to investigate the structure and interactions of a highly concentrated antibody solution over a wide range of concentrations (10-200 mg/mL) with the aid of a multivariate analysis. Our analysis of the amide I band, I 856 /I 830 of Tyr, and the relative intensity at 1004 cm(-1) of the Phe and OH stretching region at around 3000 cm(-1) showed that across this wide range of concentrations, the secondary structure of the IgG molecules did not change; however, short-range attractive interactions around the Tyr and Phe residues occurred as the distance between the IgG molecules decreased with increasing concentration. Analysis of the OH stretching region at around 3000 cm(-1) showed that these short-range attractive interactions correlated with the amount of hydrated water around the IgG molecules. Our data show that Raman spectroscopy can provide valuable information of the protein-protein interactions based on conformational approaches to support conventional colloidal approaches, especially for analyses of highly concentrated solutions.

  6. An overview of innovations and industrial solutions in Protein Microarray Technology. (United States)

    Gupta, Shabarni; Manubhai, K P; Kulkarni, Vishwesh; Srivastava, Sanjeeva


    The complexity involving protein array technology reflects in the fact that instrumentation and data analysis are subject to change depending on the biological question, technical compatibility of instruments and software used in each experiment. Industry has played a pivotal role in establishing standards for future deliberations in sustenance of these technologies in the form of protein array chips, arrayers, scanning devices, and data analysis software. This has enhanced the outreach of protein microarray technology to researchers across the globe. These have encouraged a surge in the adaptation of "nonclassical" approaches such as DNA-based protein arrays, micro-contact printing, label-free protein detection, and algorithms for data analysis. This review provides a unique overview of these industrial solutions available for protein microarray based studies. It aims at assessing the developments in various commercial platforms, thus providing a holistic overview of various modalities, options, and compatibility; summarizing the journey of this powerful high-throughput technology.

  7. Selective labeling of pulmonary surfactant protein SP-C in organic solution

    DEFF Research Database (Denmark)

    Plasencia, I; Cruz, A; López-Lacomba, J L


    Pulmonary surfactant protein SP-C has been isolated from porcine lungs and treated with dansyl isothiocyanate in chloroform:methanol 2:1 (v/v) solutions,under conditions optimized to introduce a single dansyl group covalently attached to the N-terminalamine group of the protein without loss of its...... native thioesther-linked palmitic chains. The resulting derivative Dans-SP-C conserves the secondary structure of native SP-C as well as the ability to promote interfacial adsorption of DPPC suspensions and to affect the thermotropic behavior of DPPC bilayers. This derivative can be used to characterize...... lipid-protein and protein-protein interactions of a native-like SP-C in lipid/protein complexes. Udgivelsesdato: 2001-Sep-1...

  8. Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection. (United States)

    Pridgeon, Julia W; Klesius, Phillip H; Dominowski, Paul J; Yancey, Robert J; Kievit, Michele S


    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme-g (CC-Lys-g) produced in Escherichia coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme-g plasmid DNA could be used as an immunostimulant to protect channel catfish against Aeromonas hydrophila infection. Recombinant CC-Lys-g produced in E. coli expression system exhibited significant (P recombinant channel catfish lysozyme-g (pcDNA-Lys-g) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-g offered significant (P DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P DNA injection. Taken together, our results suggest that pcDNA-Lys-g could be used as a novel immunostimulant to offer immediate protection to channel catfish against A. hydrophila infection.

  9. NMR characterization of membrane protein-detergent micelle solutions by use of microcoil equipment. (United States)

    Stanczak, Pawel; Horst, Reto; Serrano, Pedro; Wüthrich, Kurt


    Using microcoil NMR technology, the uniformly (2)H,(15)N-labeled integral membrane protein OmpX, and the phosphocholine derivative detergent Fos-10 (n-decylphosphocholine), we investigated solutions of mixed protein-detergent micelles to determine the influence of the detergent concentration on the NMR spectra of the protein. In a first step, we identified key parameters that influence the composition of the micelle solutions, which resulted in a new protocol for the preparation of well-defined concentrated protein solutions. This led to the observation that high-quality 2D [(15)N,(1)H]-transverse relaxation-optimized spectroscopy (TROSY) spectra of OmpX reconstituted in mixed micelles with Fos-10 were obtained only in a limited range of detergent concentrations. Outside of this range from about 90-180 mM, we observed a significant decrease of the average peak intensity. Relaxation-optimized NMR measurements of the rotational and translational diffusion coefficients of the OmpX/Fos-10 mixed micelles, D(r) and D(t), respectively, then showed that the stoichiometry and the effective hydrodynamic radius of the protein-containing micelles are not significantly affected by high Fos-10 concentrations and that the deterioration of NMR spectra is due to the increased viscosity at high detergent concentrations. The paper thus provides a basis for refined guidelines on the preparation of integral membrane proteins for structural studies.

  10. Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries

    Energy Technology Data Exchange (ETDEWEB)

    Pröpper, Kevin [University of Göttingen, (Germany); Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Meindl, Kathrin; Sammito, Massimo [Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Dittrich, Birger; Sheldrick, George M. [University of Göttingen, (Germany); Pohl, Ehmke, E-mail: [Durham University, (United Kingdom); Usón, Isabel, E-mail: [Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Institucio Catalana de Recerca i Estudis Avancats (ICREA), (Spain); University of Göttingen, (Germany)


    The structure solution of DNA-binding protein structures and complexes based on the combination of location of DNA-binding protein motif fragments with density modification in a multi-solution frame is described. Protein–DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein–DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite the fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein–DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein–DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.

  11. Interaction mechanism between berberine and the enzyme lysozyme (United States)

    Cheng, Ling-Li; Wang, Mei; Wu, Ming-Hong; Yao, Si-De; Jiao, Zheng; Wang, Shi-Long


    In the present paper, the interaction between model protein lysozyme (Lys) and antitumorigenic berberine (BBR) was investigated by spectroscopic methods, for finding an efficient and safe photosensitizer with highly active transient products using in photodynamic therapy study. The fluorescence data shows that the binding of BBR could change the environment of the tryptophan (Trp) residues of Lys, and form a new complex. Static quenching is the main fluorescence quenching mechanism between Lys and BBR, and there is one binding site in Lys for BBR and the type of binding force between them was determined to be hydrophobic interaction. Furthermore, the possible interaction mechanism between BBR and Lys under the photoexcitation was studied by laser flash photolysis method, the results demonstrated that BBR neutral radicals (BBR(-H)•) react with Trp (K = 3.4 × 109 M-1 s-1) via electron transfer to give the radical cation (Trp/NH•+) and neutral radical of Trp (TrpN•). Additionally BBR selectively oxidize the Trp residues of Lys was also observed by comparing the transient absorption spectra of their reaction products. Through thermodynamic calculation, the reaction mechanisms between 3BBR∗ and Trp or Lys were determined to be electron transfer process.


    Institute of Scientific and Technical Information of China (English)


    @@ A number of important methodological developments in high resolution NMR spectroscopy have led to significant increases in the size limitations that previously impeded solution structural studies of macromolecules. Specifically, isotope labeling and TROSY triple resonance spectroscopy has resulted in substantial sensitivity and resolution gain for applications to large molecular weight proteins.

  13. New insight into the solution structures of wheat gluten proteins from Raman optical activity

    DEFF Research Database (Denmark)

    Blanch, E.W.; Kasarda, D.D.; Hecht, L.


    Vibrational Raman optical activity (ROA) spectra of the wheat proteins a-gliadin (A-gliadin), omega-liadin, and a 30 kDa peptide called T-A-1 from the high molecular weight glutenin subunit (HMW-GS) Dx5 were measured to obtain new information about their solution structures. The spectral data sho...

  14. New insight into the solution structures of wheat gluten proteins from Raman optical activity

    DEFF Research Database (Denmark)

    Blanch, E.W.; Kasarda, D.D.; Hecht, L.


    Vibrational Raman optical activity (ROA) spectra of the wheat proteins a-gliadin (A-gliadin), omega-liadin, and a 30 kDa peptide called T-A-1 from the high molecular weight glutenin subunit (HMW-GS) Dx5 were measured to obtain new information about their solution structures. The spectral data sho...

  15. Theoretical aspects of pressure and solute denaturation of proteins: A Kirkwood-buff-theory approach (United States)

    Ben-Naim, Arieh


    A new approach to the problem of pressure-denaturation (PD) and solute-denaturation (SD) of proteins is presented. The problem is formulated in terms of Le Chatelier principle, and a solution is sought in terms of the Kirkwood-Buff theory of solutions. It is found that both problems have one factor in common; the excluded volumes of the folded and the unfolded forms with respect to the solvent molecules. It is shown that solvent-induced effects operating on hydrophilic groups along the protein are probably the main reason for PD. On the other hand, the SD depends on the preferential solvation of the folded and the unfolded forms with respect to solvent and co-solvent molecules.

  16. Development of a stealth carrier system for structural studies of membrane proteins in solution

    DEFF Research Database (Denmark)

    Maric, Selma

    Structural studies of membrane proteins remain a great experimental challenge. Functional reconstitution into artificial carriers that mimic the native bilayer environment allows for the handling of membrane proteins in solution and enables the use of small-angle scattering techniques for fast an......-resolution structural studes of many membrane proteins and their complexes in solution as the analysis of SANS data for this platform is greatly simplified and allows for the application of existing data analysis tools already available for soluble proteins...... and reliable structural analysis. The difficulty with this approach is that the carrier discs contribute to the measured scattering intensity in a highly non-trivial fashion, making subsequent data analysis challenging. This thesis presents the development of a specifically deuterated, stealth nanodisc system...... which can be used for SANS structural analysis of membrane proteins in solution. In combination with the D2O/H2O-based contrast variation method it is demonstrated that it is possible to prepare specifically deuterated analogues of the nanodisc, which give minimal contribution to the neutron scattering...

  17. Structures of larger proteins in solution: Three- and four-dimensional heteronuclear NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gronenborn, A.M.; Clore, G.M. [National Institutes of Health, Bethesda, MD (United States)


    Complete understanding of a protein`s function and mechanism of action can only be achieved with a knowledge of its three-dimensional structure at atomic resolution. At present, there are two methods available for determining such structures. The first method, which has been established for many years, is x-ray diffraction of protein single crystals. The second method has blossomed only in the last 5 years and is based on the application of nuclear magnetic resonance (NMR) spectroscopy to proteins in solution. This review paper describes three- and four-dimensional NMR methods applied to protein structure determination and was adapted from Clore and Gronenborn. The review focuses on the underlying principals and practice of multidimensional NMR and the structural information obtained.

  18. NMR relaxation and water self-diffusion studies in whey protein solutions and gels. (United States)

    Colsenet, Roxane; Mariette, François; Cambert, Mireille


    The changes in water proton transverse relaxation behavior induced by aggregation of whey proteins are explained in terms of the simple molecular processes of diffusion and chemical exchange. The water self-diffusion coefficient was measured in whey protein solutions and gels by the pulsed field gradient NMR method. As expected, water self-diffusion was reduced with increased protein concentrations. Whatever the concentration, the water molecules were free to diffuse over distances varying from 15 to 47 mum. Water diffusion was constant over these distances, demonstrating that no restrictions were found to explain the water hindrance. The modification in protein structure by gelation induced a decrease in water diffusion. The effects of protein concentration on water diffusion are discussed and modeled. Two approaches were compared, the obstruction effect induced by a spherical particle and the cell model, which considered two water compartments with specific self-diffusion coefficients.

  19. Molecular cloning, inducible expression and antibacterial analysis of a novel i-type lysozyme (lyz-i2) in Pacific white shrimp, Litopenaeus vannamei. (United States)

    Chen, Ting; Ren, Chunhua; Wang, Yanhong; Luo, Peng; Jiang, Xiao; Huang, Wen; Chen, Chang; Hu, Chaoqun


    The full-length cDNA coding for a novel invertebrate (i-type) lysozyme was identified in Pacific white shrimp (Litopenaeus vannamei). The newly obtained L. vannamei lysozyme is similar to the Penaeus monodon i-type lysozyme 2, but it is distant from the known L. vannamei c-type lysozyme and i-type lysozyme 1 in protein sequence; therefore, it was defined as L. vannamei i-type lysozyme 2 (lyz-i2). Expression of L. vannamei lyz-i2 transcripts were ubiquitously detected in all tissues we selected, with the highest abundance observed in the hemolymph. Challenge with Vibrio harveyi might elicit L. vannamei lyz-i2 mRNA expression in the hepatopancreas, intestine, muscle, gill and hemolymph. In the themolymph, specifically, the stimulatory effects of Vibrio and lipopolysaccharide (LPS) on lyz-i2 transcript levels were durable and transient, respectively; while Polyinosinic:polycytidylic acid [Poly (I:C)] treatment did not affect lyz-i2 expression. L. vannamei lyz-i2 recombinant protein was generated in an Escherichia coli system. By lysoplate and turbidimetric assays, the L. vannamei lyz-i2 recombinant protein showed a broad spectrum of antimicrobial properties with high activities against Micrococcaceae lysodeikticus and various Vibrio species and relatively low activity against E. coli. In conclusion, L. vannamei lyz-i2 might be a potent antibacterial protein with a role in innate immunity in Penaeid shrimp.

  20. Chaperonin-Inspired pH Protection by Mesoporous Silica SBA-15 on Myoglobin and Lysozyme. (United States)

    Lynch, Michele M; Liu, Jichuan; Nigra, Michael; Coppens, Marc-Olivier


    While enzymes are valuable tools in many fields of biotechnology, they are fragile and must be protected against denaturing conditions such as unfavorable solution pH. Within living organisms, chaperonins help enzymes fold into their native shape and protect them from damage. Inspired by this natural solution, mesoporous silica SBA-15 with different pore diameters is synthesized as a support material for immobilizing and protecting enzymes. In separate experiments, the model enzymes myoglobin and lysozyme are physically adsorbed to SBA-15 and exposed to a range of buffered pH conditions. The immobilized enzymes' biocatalytic activities are quantified and compared to the activities of nonimmobilized enzymes in the same solution conditions. It has been observed that myoglobin immobilized on SBA-15 is protected from acidic denaturation from pH 3.6 to 5.1, exhibiting relative activity of up to 350%. Immobilized lysozyme is protected from unfavorable conditions from pH 6.6 to 7.6, with relative activity of up to 200%. These results indicate that the protective effects conferred to enzymes immobilized by physical adsorption to SBA-15 are driven by the enzymes' electrostatic attraction to the material's surface. The pore diameter of SBA-15 affects the quality of protection given to immobilized enzymes, but the contribution of this effect at different pH values remains unclear.

  1. Small Angle X-Ray Scattering from Lipid-Bound Myelin Basic Protein in Solution (United States)

    Haas, H.; Oliveira, C. L. P.; Torriani, I. L.; Polverini, E.; Fasano, A.; Carlone, G.; Cavatorta, P.; Riccio, P.


    The structure of myelin basic protein (MBP), purified from the myelin sheath in both lipid-free (LF-MBP) and lipid-bound (LB-MBP) forms, was investigated in solution by small angle x-ray scattering. The water-soluble LF-MBP, extracted at pH 7.0. Under all conditions, the scattering from the two protein forms was different, indicating different molecular shapes. For the LB-MBP, well-defined scattering curves were obtained, suggesting that the protein had a unique, compact (but not globular) structure. Furthermore, these data were compatible with earlier results from molecular modeling calculations on the MBP structure which have been refined by us. In contrast, the LF-MBP data were in accordance with the expected open-coil conformation. The results represent the first direct structural information from x-ray scattering measurements on MBP in its native lipidic environment in solution. PMID:14695288

  2. Templated self-assembly of quantum dots from aqueous solution using protein scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Blum, Amy Szuchmacher [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States); Soto, Carissa M [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States); Wilson, Charmaine D [Geo-Centers, Incorporated, Newton, MA 02459 (United States); Whitley, Jessica L [Geo-Centers, Incorporated, Newton, MA 02459 (United States); Moore, Martin H [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States); Sapsford, Kim E [George Mason University, 10910 University Boulevard, Manassas, VA 20110 (United States); Lin, Tianwei [Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Chatterji, Anju [Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Johnson, John E [Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Ratna, Banahalli R [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States)


    Short, histidine-containing peptides can be conjugated to lysine-containing protein scaffolds to controllably attach quantum dots (QDs) to the scaffold, allowing for generic attachment of quantum dots to any protein without the use of specially engineered domains. This technique was used to bind quantum dots from aqueous solution to both chicken IgG and cowpea mosaic virus (CPMV), a 30 nm viral particle. These quantum dot-protein assemblies were studied in detail. The IgG-QD complexes were shown to retain binding specificity to their antigen after modification. The CPMV-QD complexes have a local concentration of quantum dots greater than 3000 nmol ml{sup -1}, and show a 15% increase in fluorescence quantum yield over free quantum dots in solution.

  3. Purification of Egg White Lysozyme from Indonesian Kampung Chicken and Ducks

    Directory of Open Access Journals (Sweden)

    Z. Wulandari


    Full Text Available Egg white lysozyme (EWL has considerably a wide functional protein exhibiting antibacterial activity mainly against Gram-positive bacteria. The EWL is widely applied in food industry and is considerably safe. Despite its high potency, EWL of Indonesian poultry has never been studied and exploited. This study was aimed to purify EWL from two Indonesian poultry: kampung chicken and Cihateup duck, and compared to egg of commercial laying hens. The eggs in this study were obtained from field laboratory of Faculty of Animal Science, Bogor Agricultural University (IPB and classified in AA quality based on the interior quality. First attempt to purify the EWL was performed by using ethanol precipitation yielding purified EWL which was still contaminated by other proteins, hence designated as partially purified EWL. Final concentrations of partially purified EWL of kampung chicken, commercial laying hens, and Cihateup duck were about 5800, 5400, and 5500 μg/mL, respectively. To confirm whether the use of ethanol in the purification affecting EWL antibacterial activities, the activities were examined against Staphylococcus aureus. It demonstrated that the partially purified EWL exhibited ability to inhibit S. aureus at 6 and 26 h suggesting that the method was feasible as it did not interfere EWL antibacterial activities. Yet, based on SDS-Page, purity was the issue in ethanol precipitation method. Further attempt using ion exchange chromatography at pH 10 successfully purified lysozyme as indicated by a single band corresponding to lysozyme size (~14 kD free from bands of other proteins. Altogether, a single step of ion exchange chromatography is sufficient and promising to isolate EWL from Indonesian poultry for various industrial purposes.

  4. Removing Endotoxin from Protein Solution by Chitosan Modified Affinity Membrane%壳聚糖亲和膜脱除蛋白质溶液中内毒素

    Institute of Scientific and Technical Information of China (English)

    孙海翔; 张林; 柴红; 陈欢林


    Affinity membrane was prepared with chitosan immobilized on the hydrophile- modified poly(vinylidene fluoride) (PVDF) membrane. Fourier transform infrared spectroscopy (FTIR) analysis indicated that the contents of-NH2 and -OH groups increased and fluoride decreased on the membrane surface after modification. Using this kind of affinity membrane, the effects of operation parameters such as pH, ionic strength and flow rate, on the amount of endotoxin removed were investigated. The results showed that the equilibrium adsorption capacity and the dissociation constant of the affinity membrane to endotoxin were 21.4 EU·mg-1 membrane and 0.50 EU·ml-1,respectively, at pH 7.0 and ionic strength 0.2 mol·L-1. Adsorption appeared to follow a typical Langmuir adsorption isotherm. At pH 5.0, ionic strength of 0.2 mol·L-1, the removal rate of endotoxin from BSA solution with the chitosan affinity membrane was up to 88.6% (11.50 EU·mg-1 membrane), and the recovery of BSA was 93.4% (0.187 mg·mg-1 membrane), while at pH 11.0, ionic strength of 0.2 mol·L-1, the removal rate of endotoxin from lysozyme solution was 72.4% (9.92 EU·mg- 1 membrane), and the recovery of lysozyme was 92.3% (0.104 mg·mg- 1 membrane).

  5. Raman mapping of mannitol/lysozyme particles produced via spray drying and single droplet drying

    DEFF Research Database (Denmark)

    Pekka Pajander, Jari; Matero, Sanni Elina; Sloth, Jakob


    PURPOSE: This study aimed to investigate the effect of a model protein on the solid state of a commonly used bulk agent in spray-dried formulations. METHODS: A series of lysozyme/mannitol formulations were spray-dried using a lab-scale spray dryer. Further, the surface temperature of drying droplet......-ray powder diffractometry (XRPD) and Raman microscopy. Partial Least Squares Discriminant Analysis was used for analyzing the Raman microscopy data. RESULTS: XRPD results indicated that a mixture of β-mannitol and α-mannitol was produced in the spray-drying process which was supported by the Raman analysis...

  6. Correlation between calculated molecular descriptors of excipient amino acids and experimentally observed thermal stability of lysozyme

    DEFF Research Database (Denmark)

    Meng-Lund, Helena; Friis, Natascha; van de Weert, Marco


    A quantitative structure-property relationship (QSPR) between protein stability and the physicochemical properties of excipients was investigated to enable a more rational choice of stabilizing excipients than prior knowledge. The thermal transition temperature and aggregation time were determined...... analysis was applied to correlate the descriptors with the experimental results. It was possible to identify descriptors, i.e. amino acids properties, with a positive influence on either transition temperature or aggregation onset time, or both. A high number of hydrogen bond acceptor moieties was the most....... The QSPR shows good correlation between calculated molecular descriptors and the measured stabilizing effect of amino acids on lysozyme....

  7. Protein-Lipid Interactions and Mechanisms of Antioxidant Activity of Proteins. (United States)


    of foods (dried, emulsified , and at natural water contents). 1) Lysozyme and peroxidizing methyl linoleate in an aqueous emulsion. 2) Lysozyme and...peroxidizing methyl linoleate in a freeze- dried system. 3) Lecithin liposomes in the presence and absence of iron and of proteins. 4) Aqueous dispersion...4 Z,. ..- : , , , , . .:: . . ,.. -. , . . . .b - ? . Egg lecithin liposomes with hen-egg lysozyme localized either inside or outside the vesicles

  8. Viscosity-Reducing Bulky-Salt Excipients Prevent Gelation of Protein, but Not Carbohydrate, Solutions. (United States)

    Kumar, Awanish; Klibanov, Alexander M


    The problem of gelation of concentrated protein solutions, which poses challenges for both downstream protein processing and liquid formulations of pharmaceutical proteins, is addressed herein by employing previously discovered viscosity-lowering bulky salts. Procainamide-HCl and the salt of camphor-10-sulfonic acid with L-arginine (CSA-Arg) greatly retard gelation upon heating and subsequent cooling of the model proteins gelatin and casein in water: Whereas in the absence of additives the proteins form aqueous gels within several hours at room temperature, procainamide-HCl for both proteins and also CSA-Arg for casein prevent gel formation for months under the same conditions. The inhibition of gelation by CSA-Arg stems exclusively from the CSA moiety: CSA-Na was as effective as CSA-Arg, while Arg-HCl was marginally or not effective. The tested bulky salts did not inhibit (and indeed accelerated) temperature-induced gel formation in aqueous solutions of all examined carbohydrates-starch, agarose, alginate, gellan gum, and carrageenan.

  9. THz time scale structural rearrangements and binding modes in lysozyme-ligand interactions. (United States)

    Woods, K N


    Predicting the conformational changes in proteins that are relevant for substrate binding is an ongoing challenge in the aim of elucidating the functional states of proteins. The motions that are induced by protein-ligand interactions are governed by the protein global modes. Our measurements indicate that the detected changes in the global backbone motion of the enzyme upon binding reflect a shift from the large-scale collective dominant mode in the unbound state towards a functional twisting deformation that assists in closing the binding cleft. Correlated motion in lysozyme has been implicated in enzyme function in previous studies, but detailed characterization of the internal fluctuations that enable the protein to explore the ensemble of conformations that ultimately foster large-scale conformational change is yet unknown. For this reason, we use THz spectroscopy to investigate the picosecond time scale binding modes and collective structural rearrangements that take place in hen egg white lysozyme (HEWL) when bound by the inhibitor (NAG)3. These protein thermal motions correspond to fluctuations that have a role in both selecting and sampling from the available protein intrinsic conformations that communicate function. Hence, investigation of these fast, collective modes may provide knowledge about the mechanism leading to the preferred binding process in HEWL-(NAG)3. Specifically, in this work we find that the picosecond time scale hydrogen-bonding rearrangements taking place in the protein hydration shell with binding modify the packing density within the hydrophobic core on a local level. These localized, intramolecular contact variations within the protein core appear to facilitate the large cooperative movements within the interfacial region separating the α- and β- domain that mediate binding. The THz time-scale fluctuations identified in the protein-ligand system may also reveal a molecular mechanism for substrate recognition.

  10. Frequency Dependent Non- Thermal Effects of Oscillating Electric Fields in the Microwave Region on the Properties of a Solvated Lysozyme System: A Molecular Dynamics Study (United States)

    Floros, Stelios; Liakopoulou-Kyriakides, Maria; Karatasos, Kostas


    The use of microwaves in every day’s applications raises issues regarding the non thermal biological effects of microwaves. In this work we employ molecular dynamics simulations to advance further the dielectric studies of protein solutions in the case of lysozyme, taking into consideration possible frequency dependent changes in the structural and dynamic properties of the system upon application of electric field in the microwave region. The obtained dielectric spectra are identical with those derived in our previous work using the Fröhlich-Kirkwood approach in the framework of the linear response theory. Noticeable structural changes in the protein have been observed only at frequencies near its absorption maximum. Concerning Cα position fluctuations, different frequencies affected different regions of the protein sequence. Furthermore, the influence of the field on the kinetics of protein-water as well as on the water-water hydrogen bonds in the first hydration shell has been studied; an extension of the Luzar-Chandler kinetic model was deemed necessary for a better fit of the applied field results and for the estimation of more accurate hydrogen bond lifetime values. PMID:28129348

  11. Explicit-water theory for the salt-specific effects and Hofmeister series in protein solutions (United States)

    Kalyuzhnyi, Yuriy V.; Vlachy, Vojko


    Effects of addition of salts on stability of aqueous protein solutions are studied theoretically and the results are compared with experimental data. In our approach, all the interacting species, proteins, ions, and water molecules, are accounted for explicitly. Water molecules are modeled as hard spheres with four off-center attractive square-well sites. These sites serve to bind either another water or to solvate the ions or protein charges. The ions are represented as charged hard spheres, and decorated by attractive sites to allow solvation. Spherical proteins simultaneously possess positive and negative groups, represented by charged hard spheres, attached to the surface of the protein. The attractive square-well sites, mimicking the protein-protein van der Waals interaction, are located on the surface of the protein. To obtain numerical results, we utilized the energy route of Wertheim's associative mean spherical approximation. From measurable properties, we choose to calculate the second virial coefficient B2, which is closely related to the tendency of proteins to aggregate and eventually crystalize. Calculations are in agreement with experimental trends: (i) For low concentration of added salt, the alkali halide salts follow the inverse Hofmeister series. (ii) At higher concentration of added salt, the trend is reversed. (iii) When cations are varied, the salts follow the direct Hofmeister series. (iv) In contrast to the colloidal theories, our approach correctly predicts the non-monotonic behavior of B2 upon addition of salts. (v) With respect to anions, the theory predicts for the B2 values to follow different sequences below and above the iso-ionic point, as also confirmed experimentally. (vi) A semi-quantitative agreement between measured and calculated values for the second virial coefficient, as functions of pH of solution and added salt type and concentration, is obtained.

  12. Effect of hydrogen peroxide on improving the heat stability of whey protein isolate solutions. (United States)

    Sutariya, Suresh; Patel, Hasmukh


    Whey protein isolate (WPI) solutions (12.8%w/w protein) were treated with varying concentrations of H2O2 in the range of 0-0.144 H2O2 to protein ratios (HTPR) by the addition of the required quantity of H2O2 and deionized water. The samples were analyzed for heat stability, rheological properties, denaturation level of β-lactoglobulin (β-LG) and α-lactalbumin (α-LA). The samples treated with H2O2 concentration >0.072 (HTPR) showed significant improvement in the heat stability, and decreased whey protein denaturation and aggregation. The WPI solution treated with H2O2 (>0.072 HTPR) remained in the liquid state after heat treatment at 120°C, whereas the control samples formed gel upon heat treatment. Detailed analysis of these samples suggested that the improvement in the heat stability of H2O2 treated WPI solution was attributed to the significant reduction in the sulfhydryl-disulfide interchange reaction during denaturation of β-LG and α-LA.

  13. Effect of the compatible solute ectoine on the stability of the membrane proteins. (United States)

    Roychoudhury, Arpita; Haussinger, Dieter; Oesterhelt, Filipp


    Mechanical single molecule techniques offer exciting possibilities for investigating protein folding and stability in native environments at sub-nanometer resolutions. Compatible solutes show osmotic activity which even at molar concentrations do not interfere with cell metabolism. They are known to protect proteins against external stress like temperature, high salt concentrations and dehydrating conditions. We studied the impact of the compatible solute ectoine (1M) on membrane proteins by analyzing the mechanical properties of Bacteriorhodopsin (BR) in its presence and absence by single molecule force spectroscopy. The unfolding experiments on BR revealed that ectoine decreases the persistence length of its polypeptide chain thereby increasing its tendency to coil up. In addition, we found higher unfolding forces indicating strengthening of those intra molecular interactions which are crucial for stability. This shows that force spectroscopy is well suited to study the effect of compatible solutes to stabilize membrane proteins against unfolding. In addition, it may lead to a better understanding of their detailed mechanism of action.

  14. Droplet evaporation of pure water and protein solution on nanostructured superhydrophobic surfaces of varying heights. (United States)

    Choi, Chang-Hwan; Kim, Chang-Jin C J


    Evaporation of liquids on substrates is important for many applications including lab-on-a-chip, especially when they are in droplets. Unlike on planar substrates, droplet evaporation on micropatterned substrates has been studied only recently and none so far on nanopatterns. Driven by the applicability of nanostructured surfaces to biomaterials and tissue engineering, we report on the evaporative process of sessile droplets of pure water and a protein solution on superhydrophobic surfaces of sharp-tip post structures in a submicrometer pitch (230 nm) and varying heights (100-500 nm). We find that the nanotopographical three-dimensionalities such as structural height and sidewall profile affect the surface superhydrophobicity in such a way that only tall and slender nanostructures provide the surface with great superhydrophobicity (a contact angle more than 170 degrees). The evaporation process was different between the pure water and the protein solution; unlike pure water, a significant contact-line spreading and pinning effect was observed in a droplet of a protein solution with an intermediate transition from a dewetting (Cassie) to a wetting (Wenzel) state. Enabled by well-defined nanostructures, our results highlight that the surface superhydrophobicity and the droplet evaporation are significantly affected by the three-dimensional nanometric topography and the surface fouling such as protein adsorption.

  15. Evaluation of activity of selected antioxidants on proteins in solution and in emulsions

    DEFF Research Database (Denmark)

    Baron, Caroline; Berner, Lis; Skibsted, L.H.


    Protection against protein oxidation by lipophilic and hydrophilic antioxidants in model systems using bovine serum albumin (BSA) in solution alone, or in an emulsion with linolenic acid methyl ester (LnMe) was found to be strongly dependent on the oxidation initiator. Tocopherol, Trolox, or the ......Protection against protein oxidation by lipophilic and hydrophilic antioxidants in model systems using bovine serum albumin (BSA) in solution alone, or in an emulsion with linolenic acid methyl ester (LnMe) was found to be strongly dependent on the oxidation initiator. Tocopherol, Trolox...... species anthracene-9,10-dipropionic acid disodium 1,4 endoperoxide (NDPO2). The results show that all the antioxidants tested were inefficient in the system with FeCl3/ascorbate. However, with the other initiating agents, the hydrophilic antioxidant, Trolox, was the most effective in preventing both...... protein and lipid oxidation. In contrast the lipophilic antioxidants were ineffective in preventing oxidation of BSA in aqueous solution, but did show some moderate antioxidative activity on protein and lipid in the BSA/LnMe system. Using the singlet oxygen generating system it was also demonstrated...

  16. Optimizing nanodiscs and bicelles for solution NMR studies of two β-barrel membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kucharska, Iga [University of Virginia, Center for Membrane Biology and Department of Molecular Physiology and Biological Physics (United States); Edrington, Thomas C. [Monsanto Company (United States); Liang, Binyong; Tamm, Lukas K., E-mail: [University of Virginia, Center for Membrane Biology and Department of Molecular Physiology and Biological Physics (United States)


    Solution NMR spectroscopy has become a robust method to determine structures and explore the dynamics of integral membrane proteins. The vast majority of previous studies on membrane proteins by solution NMR have been conducted in lipid micelles. Contrary to the lipids that form a lipid bilayer in biological membranes, micellar lipids typically contain only a single hydrocarbon chain or two chains that are too short to form a bilayer. Therefore, there is a need to explore alternative more bilayer-like media to mimic the natural environment of membrane proteins. Lipid bicelles and lipid nanodiscs have emerged as two alternative membrane mimetics that are compatible with solution NMR spectroscopy. Here, we have conducted a comprehensive comparison of the physical and spectroscopic behavior of two outer membrane proteins from Pseudomonas aeruginosa, OprG and OprH, in lipid micelles, bicelles, and nanodiscs of five different sizes. Bicelles stabilized with a fraction of negatively charged lipids yielded spectra of almost comparable quality as in the best micellar solutions and the secondary structures were found to be almost indistinguishable in the two environments. Of the five nanodiscs tested, nanodiscs assembled from MSP1D1ΔH5 performed the best with both proteins in terms of sample stability and spectral resolution. Even in these optimal nanodiscs some broad signals from the membrane embedded barrel were severely overlapped with sharp signals from the flexible loops making their assignments difficult. A mutant OprH that had two of the flexible loops truncated yielded very promising spectra for further structural and dynamical analysis in MSP1D1ΔH5 nanodiscs.

  17. Invertebrate lysozymes: Diversity and distribution, molecular mechanism and in vivo function

    Indian Academy of Sciences (India)

    Joris M Van Herreweghe; Chris W Michiels


    Lysozymes are antibacterial enzymes widely distributed among organisms. Within the animal kingdom, mainly three major lysozyme types occur. Chicken (c)-type lysozyme and goose (g)-type lysozyme are predominantly, but not exclusively, found in vertebrate animals, while the invertebrate (i)-type lysozyme is typical for invertebrate organisms, and hence its name. Since their discovery in 1975, numerous research articles report on the identification of i-type lysozymes in a variety of invertebrate phyla. This review describes the current knowledge on i-type lysozymes, outlining their distribution, molecular mechanism and in vivo function taking the representative from Venerupis philippinarum (formerly Tapes japonica) (Vp-ilys) as a model. In addition, invertebrate g-type and ch-type (chalaropsis) lysozymes, which have been described in molluscs and nematodes, respectively, are also briefly discussed.

  18. Lysozyme refolding at high concentration by dilution and size—exclusion chromatography

    Institute of Scientific and Technical Information of China (English)

    高永贵; 关怡新; 姚善泾; CHOMan-gi


    This study of renaturation by dilution and size exclusion chromstography(SEC) addition of urea to improve yield as well as the initial and final protein concentrations showed that although urea decreased the rate of lysozyme refolding,it could suppress protein aggregation to sustain the pathway of correct refolding at high protein concentration;and that there existed an optimum urea concentration in renaturation buffer.Under the above conditions,lysozyme was successfully refolded from initial concentration of up tp 40 mg/mL by dilu-tion and 100 mg/mL by SEC,with the yield of the former being more than 40% and that of the latter being 34.8%.Especislly,under the condition of 30 min iterval time,i.e.τ>2(tR2-tR1),the efficiency was increased by 25% and the renaturation buffer could be recycled for SEC refolding in continuous operation of downstream process.

  19. Curcumin and kaempferol prevent lysozyme fibril formation by modulating aggregation kinetic parameters. (United States)

    Borana, Mohanish S; Mishra, Pushpa; Pissurlenkar, Raghuvir R S; Hosur, Ramakrishna V; Ahmad, Basir


    Interaction of small molecule inhibitors with protein aggregates has been studied extensively, but how these inhibitors modulate aggregation kinetic parameters is little understood. In this work, we investigated the ability of two potential aggregation inhibiting drugs, curcumin and kaempferol, to control the kinetic parameters of aggregation reaction. Using thioflavin T fluorescence and static light scattering, the kinetic parameters such as amplitude, elongation rate constant and lag time of guanidine hydrochloride-induced aggregation reactions of hen egg white lysozyme were studied. We observed a contrasting effect of inhibitors on the kinetic parameters when aggregation reactions were measured by these two probes. The interactions of these inhibitors with hen egg white lysozyme were investigated using fluorescence quench titration method and molecular dynamics simulations coupled with binding free energy calculations. We conclude that both the inhibitors prolong nucleation of amyloid aggregation through binding to region of the protein which is known to form the core of the protein fibril, but once the nucleus is formed the rate of elongation is not affected by the inhibitors. This work would provide insight into the mechanism of aggregation inhibition by these potential drug molecules. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. High-Pressure-High-Temperature Processing Reduces Maillard Reaction and Viscosity in Whey Protein-Sugar Solutions

    NARCIS (Netherlands)

    Avila Ruiz, Geraldine; Xi, Bingyan; Minor, Marcel; Sala, Guido; Boekel, van Tiny; Fogliano, Vincenzo; Stieger, Markus


    The aim of the study was to determine the influence of pressure in high-pressure–high-temperature (HPHT) processing on Maillard reactions and protein aggregation of whey protein–sugar solutions. Solutions of whey protein isolate containing either glucose or trehalose at pH 6, 7, and 9 were treated

  1. Solution-based functionalization of gallium nitride nanowires for protein sensor development (United States)

    Williams, Elissa H.; Davydov, Albert V.; Oleshko, Vladimir P.; Steffens, Kristen L.; Levin, Igor; Lin, Nancy J.; Bertness, Kris A.; Manocchi, Amy K.; Schreifels, John A.; Rao, Mulpuri V.


    A solution-based functionalization method for the specific and selective attachment of the streptavidin (SA) protein to gallium nitride (GaN) nanowires (NWs) is presented. By exploiting streptavidin's strong affinity for its ligand biotin, SA immobilization on GaN NWs was achieved by exposing the GaN NW surface to a 3-aminopropyltriethoxysilane (APTES) solution followed by reaction with biotin. Functionalization of the NWs with APTES was facilitated by the presence of an ≈ 1 nm thick surface oxide layer, which formed on the NWs after exposure to air and oxygen plasma. Biotinylation was accomplished by reacting the APTES-functionalized NWs with sulfo-N-hydroxysuccinimide-biotin at slightly alkaline pH. It was determined that the biotinylated GaN NW surface was specific towards the binding of SA and demonstrated no affinity towards a control protein, bovine serum albumin (BSA). There was however, evidence of non-specific, electrostatic binding of both the SA protein and the BSA protein to the APTES-coated NWs, revealing the importance of the biotinylation step. Successful SA immobilization on the biotinylated GaN NW surface was verified using fluorescence microscopy, field-emission scanning electron microscopy, high-resolution transmission electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. The functionalized GaN NWs demonstrate potential as biosensing platforms for the selective detection of proteins.

  2. Molecular dynamics simulation of triclinic lysozyme in a crystal lattice. (United States)

    Janowski, Pawel A; Liu, Chunmei; Deckman, Jason; Case, David A


    Molecular dynamics simulations of crystals can enlighten interpretation of experimental X-ray crystallography data and elucidate structural dynamics and heterogeneity in biomolecular crystals. Furthermore, because of the direct comparison against experimental data, they can inform assessment of molecular dynamics methods and force fields. We present microsecond scale results for triclinic hen egg-white lysozyme in a supercell consisting of 12 independent unit cells using four contemporary force fields (Amber ff99SB, ff14ipq, ff14SB, and CHARMM 36) in crystalline and solvated states (for ff14SB only). We find the crystal simulations consistent across multiple runs of the same force field and robust to various solvent equilibration schemes. However, convergence is slow compared with solvent simulations. All the tested force fields reproduce experimental structural and dynamic properties well, but Amber ff14SB maintains structure and reproduces fluctuations closest to the experimental model: its average backbone structure differs from the deposited structure by 0.37Å; by contrast, the average backbone structure in solution differs from the deposited by 0.65Å. All the simulations are affected by a small progressive deterioration of the crystal lattice, presumably due to imperfect modeling of hydrogen bonding and other crystal contact interactions; this artifact is smallest in ff14SB, with average lattice positions deviating by 0.20Å from ideal. Side-chain disorder is surprisingly low with fewer than 30% of the nonglycine or alanine residues exhibiting significantly populated alternate rotamers. Our results provide helpful insight into the methodology of biomolecular crystal simulations and indicate directions for future work to obtain more accurate energy models for molecular dynamics.

  3. Purification, amino acid sequence, and some properties of rabbit kidney lysozyme. (United States)

    Ito, Y; Yamada, H; Nakamura, S; Imoto, T


    The lysozyme (rabbit kidney lysozyme) from the homogenate of rabbit kidney (Japanese white) was purified by repeated cation-exchange chromatography on Bio-Rex 70. The amino acid sequence was determined by automated gas-phase Edman degradation of the peptides obtained from the digestion of reduced and S-carboxymethylated rabbit lysozyme with Achromobacter protease I (lysyl endopeptidase). The sequence thus determined was KIYERCELARTLKKLGLDGYKGVSLANWMCLAKWESSYNTRATNYNPGDKSTDYGIFQ INSRYWCNDGKTPRAVNACHIPCSDLLKDDITQAVACAKRVVSDPQGIRAWVAWRNHCQ NQDLTPYIRGCGV, indicating 25 amino acid substitutions from human lysozyme. The lytic activity of rabbit lysozyme against Micrococcus lysodeikticus at pH 7, ionic strength of 0.1, and 30 degrees C was found to be 190 and 60% of those of hen and human lysozymes, respectively. The lytic activity-pH profile of rabbit lysozyme was slightly different from those of hen and human lysozymes. While hen and human lysozymes had wide optimum activities at around pH 5.5-8.5, the optimum activity of rabbit lysozyme was at around pH 5.5-7.0. The high proline content (five residues per molecule compared with two prolines per molecule in hen or human lysozyme) is one of the interesting features of rabbit lysozyme. The transition temperatures for the unfolding of rabbit, human, and hen lysozymes in 3 M guanidine hydrochloride at pH 5.5 were 51.2, 45.5, and 45.4 degrees C, respectively, indicating that rabbit lysozyme is stabler than the other two lysozymes. The high proline content may be responsible for the increased stability of rabbit lysozyme.

  4. Immunohistochemical localization of human immunoglobulins and lysozyme in epoxy-embedded lymph nodes: effect of different fixatives and of proteolytic digestion. (United States)

    Dell'Orto, P; Viale, G; Colombi, R; Braidotti, P; Coggi, G


    The postembedding immunoperoxidase staining technique for the localization of immunoglobulins (light and heavy chains) and of lysozyme has been successfully applied to epoxy-embedded human lymph nodes, after removal of the resin. Glutaraldehyde-containing fixatives appear to be suitable for the immunohistochemical localization of human immunoglobulins and lysozyme, provided that the masked antigenicity of these proteins is recovered by proteolytic digestion of the tissue sections using 0.4% pepsin or 0.1% trypsin. Nonglutaraldehyde-containing fixatives allow the immunolocalization of human immunoglobulins without any enzymatic pretreatment. This study shows that tissues routinely fixed in glutaraldehyde and embedded for ultrastructural investigations are actually suitable for immunohistochemical studies on human immunoglobulins and lysozyme.

  5. Effect of ethanol-water mixture on the structure and dynamics of lysozyme: A fluorescence correlation spectroscopy study (United States)

    Chattoraj, Shyamtanu; Mandal, Amit Kumar; Bhattacharyya, Kankan


    Effect of ethanol-water mixture on the hydrodynamic radius (rH) and conformational dynamics of lysozyme has been studied by circular dichroism, emission spectra, and fluorescence correlation spectroscopy. For this purpose, the protein lysozyme is covalently labeled near the active site with a fluorescent probe, alexa 488. The ethanol molecules are sequestered near the hydrophobic tryptophan residues as indicated by the blue shift of the emission maximum of tryptophan. It is observed that both size (rH) and time constant of conformational relaxation (τR) of lysozyme oscillate with increase in ethanol concentration. The rH of the protein fluctuates from 19 Å in the native state, to a minimum of 13 Å, and a maximum of 29 Å. It is proposed that the oscillating behavior arises from competition between mutual interaction among protein, ethanol, and water. The fluorescence intensity fluctuates because of quenching of the fluorescence of the probe (alexa) by the free amino group of certain residues (e.g., tryptophan). Rate of inter-conversion (folding dynamics) between the open (fluorescent) and closed (non-fluorescent) form has been determined and is found to exhibit similar oscillation with variation in ethanol content.

  6. Fluorescence detection of lipid-induced oligomeric intermediates involved in lysozyme "amyloid-like" fiber formation driven by anionic membranes. (United States)

    Melo, Ana M; Ricardo, Joana C; Fedorov, Aleksander; Prieto, Manuel; Coutinho, Ana


    Recent findings implicate that "amyloid-like" fiber formation by several non-amyloidogenic proteins/peptides can be triggered by negatively charged lipid membranes. In order to elucidate the factors that govern the formation of these structures, the interaction of lysozyme with phosphatidylserine-containing lipid vesicles was studied by steady-state and time-resolved fluorescence measurements. Three consecutive stages in the interaction of Alexa488-fluorescently labeled lysozyme (Lz-A488) with acidic lipid vesicles were identified in ensemble average measurements. The variation of the mean fluorescence lifetime of Lz-A488 as a function of the surface coverage of the liposomes was quantitatively described by a cooperative partition model that assumes that monomeric lysozyme molecules partition into the bilayer surface and reversibly assemble into oligomers with k subunits (k ≥ 6). The global fit to the experimental data covering a wide range of experimental conditions was performed by taking into account electrostatic effects by means of the Gouy-Chapman theory using a single self-consistent pair of parameters (aggregation constant and stoichiometry). The lipid-protein supramolecular assemblies formed at a low lipid/protein molar ratio were further characterized by fluorescence lifetime imaging microscopy at the single-fiber level, which reported that quenched oligomers are the predominant species in these structures.

  7. Stealth carriers for low-resolution structure determination of membrane proteins in solution

    DEFF Research Database (Denmark)

    Maric, Selma; Skar-Gislinge, Nicholas; Midtgaard, Søren


    Structural studies of membrane proteins remain a great experimental challenge. Functional reconstitution into artificial nanoscale bilayer disc carriers that mimic the native bilayer environment allows the handling of membrane proteins in solution. This enables the use of small-angle scattering...... techniques for fast and reliable structural analysis. The difficulty with this approach is that the carrier discs contribute to the measured scattering intensity in a highly nontrivial fashion, making subsequent data analysis challenging. Here, an elegant solution to circumvent the intrinsic complexity...... brought about by the presence of the carrier disc is presented. In combination with small-angle neutron scattering (SANS) and the D2O/H2O-based solvent contrast-variation method, it is demonstrated that it is possible to prepare specifically deuterated carriers that become invisible to neutrons in 100% D2...

  8. Solution structure of an archaeal DNA binding protein with an eukaryotic zinc finger fold.

    Directory of Open Access Journals (Sweden)

    Florence Guillière

    Full Text Available While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers.

  9. Proteins in frozen solutions: evidence of ice-induced partial unfolding.


    Strambini, G B; Gabellieri, E


    From a drastic decrease in the phosphorescence lifetime of tryptophan residues buried in compact rigid cores of globular proteins, it was possible to demonstrate that freezing of aqueous solutions is invariably accompanied by a marked loosening of the native fold, an alteration that entails considerable loss of secondary and tertiary structure. The phenomenon is largely reversible on ice melting although, in some cases, a small fraction of macromolecules recovers neither the initial phosphore...

  10. Using in situ X-ray reflectivity to study protein adsorption on hydrophilic and hydrophobic surfaces: benefits and limitations. (United States)

    Richter, Andrew G; Kuzmenko, Ivan


    We have employed in situ X-ray reflectivity (IXRR) to study the adsorption of a variety of proteins (lysozyme, cytochrome c, myoglobin, hemoglobin, serum albumin, and immunoglobulin G) on model hydrophilic (silicon oxide) and hydrophobic surfaces (octadecyltrichlorosilane self-assembled monolayers), evaluating this recently developed technique for its applicability in the area of biomolecular studies. We report herein the highest resolution depiction of adsorbed protein films, greatly improving on the precision of previous neutron reflectivity (NR) results and previous IXRR studies. We were able to perform complete scans in 5 min or less with the maximum momentum transfer of at least 0.52 Å(-1), allowing for some time-resolved information about the evolution of the protein film structure. The three smallest proteins (lysozyme, cytochrome c, and myoglobin) were seen to deposit as fully hydrated, nondenatured molecules onto hydrophilic surfaces, with indications of particular preferential orientations. Time evolution was observed for both lysozyme and myoglobin films. The larger proteins were not observed to deposit on the hydrophilic substrates, perhaps because of contrast limitations. On hydrophobic surfaces, all proteins were seen to denature extensively in a qualitatively similar way but with a rough trend that the larger proteins resulted in lower coverage. We have generated high-resolution electron density profiles of these denatured films, including capturing the growth of a lysozyme film. Because the solution interface of these denatured films is diffuse, IXRR cannot unambiguously determine the film extent and coverage, a drawback compared to NR. X-ray radiation damage was systematically evaluated, including the controlled exposure of protein films to high-intensity X-rays and exposure of the hydrophobic surface to X-rays before adsorption. Our analysis showed that standard measuring procedures used for XRR studies may lead to altered protein films

  11. Effect of the environment on the protein dynamical transition: a neutron scattering study. (United States)

    Paciaroni, Alessandro; Cinelli, Stefania; Onori, Giuseppe


    We performed an elastic neutron scattering investigation of the molecular dynamics of lysozyme solvated in glycerol, at different water contents h (grams of water/grams of lysozyme). The marked non-Gaussian behavior of the elastic intensity was studied in a wide experimental momentum transfer range, as a function of the temperature. The internal dynamics is well described in terms of the double-well jump model. At low temperature, the protein total mean square displacements exhibit an almost linear harmonic trend irrespective of the hydration level, whereas at the temperature T(d) a clear changeover toward an anharmonic regime marks a protein dynamical transition. The decrease of T(d) from approximately 238 K to approximately 195 K as a function of h is reminiscent of that found in the glass transition temperature of aqueous solutions of glycerol, thus suggesting that the protein internal dynamics as a whole is slave to the environment properties. Both T(d) and the total mean square displacements indicate that the protein flexibility strongly rises between 0.1 and 0.2h. This hydration-dependent dynamical activation, which is similar to that of hydrated lysozyme powders, is related to the specific interplay of the protein with the surrounding water and glycerol molecules.

  12. Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species. (United States)

    Hikima, Sonomi; Hikima, Jun ichi; Rojtinnakorn, Jiraporn; Hirono, Ikuo; Aoki, Takashi


    Lysozyme cDNA was isolated from a kuruma shrimp, Marsupenaeus japonicus, hemocyte cDNA library. The cDNA consists of 1055 base pairs (bp) and encodes a chicken-type (c-type) lysozyme with a deduced amino acid sequence of 156 residues. The kuruma shrimp lysozyme has a high identity (79.7%) with pacific white shrimp lysozyme, and low to moderate identities (33.3-43.0%) with lysozymes of insects and vertebrates. Comparisons with other c-type lysozymes from invertebrates and vertebrates showed that the two catalytic residues (Glu58 and Asp75) and the eight cysteine residue motif were completely conserved. Two novel insertion sequences were also observed in the kuruma and pacific white shrimp lysozyme amino acid sequences. Interestingly, phylogenetic analysis revealed that the kuruma shrimp lysozyme was more closely related to vertebrate c-type lysozymes. Expression of the cDNA in insect cells, using a baculovirus expression system, yielded a recombinant lysozyme with optimum activity at pH 7.5 and 50 degrees C, as evaluated by a lysoplate assay. The kuruma shrimp lysozyme displayed lytic activities against several Vibrio species and fish pathogens, including Vibrio penaeicida (a pathogenic bacteria to the kuruma shrimp) and suggested that shrimp lysozyme affects a greater variety of pathogens.

  13. A Comparison of Blood Factor XII Autoactivation in Buffer, Protein Cocktail, Serum, and Plasma Solutions (United States)

    Golas, Avantika; Yeh, Chyi-Huey Josh; Pitakjakpipop, Harit; Siedlecki, Christopher A.; Vogler, Erwin A.


    Activation of blood plasma coagulation in vitro by contact with material surfaces is demonstrably dependent on plasma-volume-to-activator-surface-area ratio. The only plausible explanation consistent with current understanding of coagulation-cascade biochemistry is that procoagulant stimulus arising from the activation complex of the intrinsic pathway is dependent on activator surface area. And yet, it is herein shown that activation of the blood zymogen factor XII (Hageman factor, FXII) dissolved in buffer, protein cocktail, heat-denatured serum, and FXI deficient plasma does not exhibit activator surface-area dependence. Instead, a highly-variable burst of procoagulant-enzyme yield is measured that exhibits no measurable kinetics, sensitivity to mixing, or solution-temperature dependence. Thus, FXII activation in both buffer and protein-containing solutions does not exhibit characteristics of a biochemical reaction but rather appears to be a “mechanochemical” reaction induced by FXII molecule interactions with hydrophilic activator particles that do not formally adsorb blood proteins from solution. Results of this study strongly suggest that activator surface-area dependence observed in contact activation of plasma coagulation does not solely arise at the FXII activation step of the intrinsic pathway. PMID:23117212

  14. NMR study of a membrane protein in detergent-free aqueous solution. (United States)

    Zoonens, Manuela; Catoire, Laurent J; Giusti, Fabrice; Popot, Jean-Luc


    One of the major obstacles to membrane protein (MP) structural studies is the destabilizing effect of detergents. Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep MPs water-soluble under mild conditions. In the present work, we have explored the feasibility of studying the structure of APol-complexed MPs by NMR. As a test MP, we chose the 171-residue transmembrane domain of outer MP A from Escherichia coli (tOmpA), whose x-ray and NMR structures in detergent are known. 2H,15N-labeled tOmpA was produced as inclusion bodies, refolded in detergent solution, trapped with APol A8-35, and the detergent removed by adsorption onto polystyrene beads. The resolution of transverse relaxation-optimized spectroscopy-heteronuclear single-quantum correlation spectra of tOmpA/A8-35 complexes was found to be close to that of the best spectra obtained in detergent solutions. The dispersion of chemical shifts indicated that the protein had regained its native fold and retained it during the exchange of surfactants. MP-APol interactions were mapped by substituting hydrogenated for deuterated A8-35. The resulting dipolar broadening of amide proton linewidths was found to be limited to the beta-barrel region of tOmpA, indicating that A8-35 binds specifically to the hydrophobic transmembrane surface of the protein. The potential of this approach to MP studies by solution NMR is discussed.

  15. Antimicrobial chitosan-lysozyme (CL) films and coatings for enhancing microbial safety of mozzarella cheese. (United States)

    Duan, J; Park, S-I; Daeschel, M A; Zhao, Y


    This study investigated the antimicrobial activities of chitosan-lysozyme (CL) composite films and coatings against tested microorganisms inoculated onto the surface of Mozzarella cheese. CL film-forming solutions (FFS) with a pH of 4.4 to 4.5 were prepared by incorporating 0% or 60% lysozyme (per dry weight of chitosan) into chitosan FFS with or without a pH adjustment to 5.2. Sliced cheese was subjected to 3 CL package applications: film, lamination on a multilayer coextruded film, and coating. Cheese was inoculated with Listeria monocytogenes, Escherichia coli, or Pseudomonas fluorescens at 10(4) CFU/g, or with mold and yeast at 10(2) CFU/g. Inoculated cheese was individually vacuum packaged and stored at 10 degrees C for sampling at 1, 7, and 14 d for bacteria, and at 10, 20, and 30 d for fungi. Inoculated bacteria survived but failed to multiply in untreated cheese during storage. Treated cheese received 0.43- to 1.25-, 0.40- to 1.40-, and 0.32- to 1.35-log reductions in E. coli, P. fluorescens, and L. monocytogenes, respectively. Incorporation of 60% lysozyme in chitosan FFS showed greater antimicrobial effect than chitosan alone on P. fluorescens and L. monocytogenes. The pH adjustment only affected the antimicrobial activity on L. monocytogenes, with lower pH (unadjusted) showing greater antimicrobial effect than pH 5.2. Mold and yeast increased to 10(5) CFU/g in untreated cheese after 30 d storage. Growth of mold was completely inhibited in cheese packaged with CL films, while 0.24- to 1.90- and 0.06- to 0.50-log reductions in mold populations were observed in cheese packaged with CL-laminated films and coatings, respectively. All CL packaging applications resulted in 0.01- to 0.64-log reduction in yeast populations.

  16. Production of human lactoferrin and lysozyme in the milk of transgenic dairy animals: past, present, and future. (United States)

    Cooper, Caitlin A; Maga, Elizabeth A; Murray, James D


    Genetic engineering, which was first developed in the 1980s, allows for specific additions to animals' genomes that are not possible through conventional breeding. Using genetic engineering to improve agricultural animals was first suggested when the technology was in the early stages of development by Palmiter et al. (Nature 300:611-615, 1982). One of the first agricultural applications identified was generating transgenic dairy animals that could produce altered or novel proteins in their milk. Human milk contains high levels of antimicrobial proteins that are found in low concentrations in the milk of ruminants, including the antimicrobial proteins lactoferrin and lysozyme. Lactoferrin and lysozyme are both part of the innate immune system and are secreted in tears, mucus, and throughout the gastrointestinal (GI) tract. Due to their antimicrobial properties and abundance in human milk, multiple lines of transgenic dairy animals that produce either human lactoferrin or human lysozyme have been developed. The focus of this review is to catalogue the different lines of genetically engineered dairy animals that produce either recombinant lactoferrin or lysozyme that have been generated over the years as well as compare the wealth of research that has been done on the in vitro and in vivo effects of the milk they produce. While recent advances including the development of CRISPRs and TALENs have removed many of the technical barriers to predictable and efficient genetic engineering in agricultural species, there are still many political and regulatory hurdles before genetic engineering can be used in agriculture. It is important to consider the substantial amount of work that has been done thus far on well established lines of genetically engineered animals evaluating both the animals themselves and the products they yield to identify the most effective path forward for future research and acceptance of this technology.

  17. Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis

    DEFF Research Database (Denmark)

    Leon, Ileana R; Schwämmle, Veit; Jensen, Ole N;


    a combination of qualitative and quantitative LC-MS/MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein...... conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents prior to analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative LC-MS/MS workflow quantified over 3700 distinct peptides with 96% completeness between all...... protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows...

  18. Label-free free-solution nanoaperture optical tweezers for single molecule protein studies. (United States)

    Al Balushi, Ahmed A; Kotnala, Abhay; Wheaton, Skyler; Gelfand, Ryan M; Rajashekara, Yashaswini; Gordon, Reuven


    Nanoaperture optical tweezers are emerging as useful label-free, free-solution tools for the detection and identification of biological molecules and their interactions at the single molecule level. Nanoaperture optical tweezers provide a low-cost, scalable, straight-forward, high-speed and highly sensitive (SNR ∼ 33) platform to observe real-time dynamics and to quantify binding kinetics of protein-small molecule interactions without the need to use tethers or labeling. Such nanoaperture-based optical tweezers, which are 1000 times more efficient than conventional optical tweezers, have been used to trap and isolate single DNA molecules and to study proteins like p53, which has been claimed to be in mutant form for 75% of human cancers. More recently, nanoaperture optical tweezers have been used to probe the low-frequency (in the single digit wavenumber range) Raman active modes of single nanoparticles and proteins. Here we review recent developments in the field of nanoaperture optical tweezers and how they have been applied to protein-antibody interactions, protein-small molecule interactions including single molecule binding kinetics, and protein-DNA interactions. In addition, recent works on the integration of nanoaperture optical tweezers at the tip of optical fiber and in microfluidic environments are presented.

  19. Carnosine's effect on amyloid fibril formation and induced cytotoxicity of lysozyme.

    Directory of Open Access Journals (Sweden)

    Josephine W Wu

    Full Text Available Carnosine, a common dipeptide in mammals, has previously been shown to dissemble alpha-crystallin amyloid fibrils. To date, the dipeptide's anti-fibrillogensis effect has not been thoroughly characterized in other proteins. For a more complete understanding of carnosine's mechanism of action in amyloid fibril inhibition, we have investigated the effect of the dipeptide on lysozyme fibril formation and induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Our study demonstrates a positive correlation between the concentration and inhibitory effect of carnosine against lysozyme fibril formation. Molecular docking results show carnosine's mechanism of fibrillogenesis inhibition may be initiated by binding with the aggregation-prone region of the protein. The dipeptide attenuates the amyloid fibril-induced cytotoxicity of human neuronal cells by reducing both apoptotic and necrotic cell deaths. Our study provides solid support for carnosine's amyloid fibril inhibitory property and its effect against fibril-induced cytotoxicity in SH-SY5Y cells. The additional insights gained herein may pave way to the discovery of other small molecules that may exert similar effects against amyloid fibril formation and its associated neurodegenerative diseases.

  20. Role of Molecular Flexibility and Colloidal Descriptions of Proteins in Crowded Environments from Small-Angle Scattering. (United States)

    Castellanos, Maria Monica; Clark, Nicholas J; Watson, Max C; Krueger, Susan; McAuley, Arnold; Curtis, Joseph E


    Small-angle scattering is a powerful technique to study molecular conformation and interactions of proteins in solution and in amorphous solids. We have investigated the role of multiple protein configurations in the interaction parameters derived from small-angle scattering for proteins in concentrated solutions. In order to account for the wide configurational space sampled by proteins, we generate ensembles of atomistic structures for lysozyme and monoclonal antibodies, representing globular and flexible proteins, respectively. While recent work has argued that a colloidal approach is inadequate to model proteins, because of the large configurational space that they sample in solution, we find a range of length scales where colloidal models can be used to describe solution scattering data while simultaneously accounting for structural flexibility. We provide insights to determine the length scales where isotropic colloidal models can be used, and find smoothly varying sets of interaction parameters that encompass ensembles of structures. This approach may play an important role in the definition of long-range interactions in coarse-grained models of flexible proteins with experimental scattering constraints. Additionally, we apply the decoupling approximation to ensembles of lysozyme structures with atomistic detail and observe remarkably different results when using geometric solids, such as ellipsoids. The insights from this study provide guidelines for the analysis of small-angle scattering profiles of proteins in crowded environments.

  1. High-Pressure-High-Temperature Processing Reduces Maillard Reaction and Viscosity in Whey Protein-Sugar Solutions. (United States)

    Avila Ruiz, Geraldine; Xi, Bingyan; Minor, Marcel; Sala, Guido; van Boekel, Martinus; Fogliano, Vincenzo; Stieger, Markus


    The aim of the study was to determine the influence of pressure in high-pressure-high-temperature (HPHT) processing on Maillard reactions and protein aggregation of whey protein-sugar solutions. Solutions of whey protein isolate containing either glucose or trehalose at pH 6, 7, and 9 were treated by HPHT processing or conventional high-temperature (HT) treatments. Browning was reduced, and early and advanced Maillard reactions were retarded under HPHT processing at all pH values compared to HT treatment. HPHT induced a larger pH drop than HT treatments, especially at pH 9, which was not associated with Maillard reactions. After HPHT processing at pH 7, protein aggregation and viscosity of whey protein isolate-glucose/trehalose solutions remained unchanged. It was concluded that HPHT processing can potentially improve the quality of protein-sugar-containing foods, for which browning and high viscosities are undesired, such as high-protein beverages.

  2. Functional Biomaterials: Solution Electrospinning and Gelation of Whey Protein and Pullulan (United States)

    Sullivan, Stephanie Tolstedt

    Utilizing biomaterials that are biodegradable, biocompatible and edible serve well for food products as well as biomedical applications. Biomaterials whey protein and pullulan both have these characteristics. Whey proteins (WP) have been used in food products for many years and more recently in pharmaceutical products. They have the ability to form both gels and stable foams. Pullulan (PULL) has also been used in both food and pharmaceutical products, and is a highly water soluble, non-gelling polysaccharide and has been used primarily as a film former. Herein, we investigate the ability of whey protein and pullulan to form nanofibers and gels. Combining their distinct properties allows the ability to uniquely manipulate nanofiber and gel characteristics and behavior for a variety of applications, from food to even tissue scaffolding. First, we determined the electrospinnability of aqueous whey protein solutions. Both whey protein isolate (WPI) and one of its major components beta--lactoglobulin (BLG), either in native or denatured form, yielded interesting micro and nanostructures when electrosprayed; while nanofiber production required blending with a spinnable polymer, poly(ethylene oxide) (PEO). WP:PEO solutions were also successfully electrospun at acidic pH (2≤pH≤3), which could improve shelf life. Fourier Transform Infrared Reflectance (FTIR) analysis of WP:PEO fiber mat indicated some variation in WP secondary structure with varying WPI concentration (as WPI increased, % alpha-helix increased and beta-turn decreased) and pH (as pH decreased from neutral (7.5) to acidic (2), % beta-sheet decreased and alpha-helix increased). X-ray Photoelectron Spectroscopy (XPS) also confirmed the presence of WP on the surface of the blend fibers, augmenting the FTIR analysis. Interestingly, WP:PEO composite nanofibers maintained its fibrous morphology at temperatures as high as 100 °C, above the 60 °C PEO melting point. Further, we show that the blend mats retained a

  3. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

    Energy Technology Data Exchange (ETDEWEB)

    Lee, A L [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry


    Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the {delta}-Al-{var_epsilon} activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a {beta}{alpha}{beta}-{beta}{alpha}{beta} pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel {beta}-sheet. In addition {sup 15}N T{sub 1}, T{sub 2}, and {sup 15}N/{sup 1}H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone {sup 1}H, {sup 13}C, and {sup 15}N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and {sup 15}N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.

  4. Novel approach to protein crystallizations: Control of the phase behavior of aqueous solutions using microfluidics (United States)

    Shim, Jung Uk

    A microfluidic device denoted the Phase Chip has been developed to exploit the permeation of water through poly(dimethylsiloxane) (PDMS) in order to vary the concentration of aqueous nanoliter volume microdrops stored in wells. The permeation of water in the Phase Chip is modeled using the diffusion equation and good agreement between experiment and theory is obtained. The phase diagram of a polymer/salt mixture is measured employing the Phase Chip and agrees well with the phase diagram obtained off-chip. The Phase Chip first creates drops of the polymer/salt mixture whose composition varies sequentially. Subsequently the drops are docked in storage wells and the concentration of each stored drop is controlled by varying the water activity of a reservoir that is separated from the drops by a thin layer of PDMS through which water, but not the solutes, permeates. The Phase Chip, incorporating a dialysis membrane on-chip, presents several advantages for protein crystallizations. First, protein crystallization is a non-equilibrium process so it makes sense to have dynamic control over the key thermodynamic variable; concentration. The Phase Chip, with its ability to reversibly control protein and precipitant concentrations, renders varying concentration as convenient as varying temperature. Second, by varying the water content of each drop we can explore many different crystallization conditions in the same drop. Finally, we have demonstrated that we can first formulate stable protein solutions, next induce nucleation and then grow large protein crystals. For these reasons, the Phase Chip promises to be a faster, better, and cheaper method for protein crystallization.

  5. Two goose-type lysozymes in Mytilus galloprovincialis: possible function diversification and adaptive evolution.

    Directory of Open Access Journals (Sweden)

    Qing Wang

    Full Text Available Two goose-type lysozymes (designated as MGgLYZ1 and MGgLYZ2 were identified from the mussel Mytilus galloprovincialis. MGgLYZ1 mRNA was widely expressed in the examined tissues and responded sensitively to bacterial challenge in hemocytes, while MGgLYZ2 mRNA was predominately expressed and performed its functions in hepatopancreas. However, immunolocalization analysis showed that both these lysozymes were expressed in all examined tissues with the exception of adductor muscle. Recombinant MGgLYZ1 and MGgLYZ2 could inhibit the growth of several Gram-positive and Gram-negative bacteria, and they both showed the highest activity against Pseudomonas putida with the minimum inhibitory concentration (MIC of 0.95-1.91 µM and 1.20-2.40 µM, respectively. Protein sequences analysis revealed that MGgLYZ2 had lower isoelectric point and less protease cutting sites than MGgLYZ1. Recombinant MGgLYZ2 exhibited relative high activity at acidic pH of 4-5, while MGgLYZ1 have an optimum pH of 6. These results indicated MGgLYZ2 adapted to acidic environment and perhaps play an important role in digestion. Genomic structure analysis suggested that both MGgLYZ1 and MGgLYZ2 genes are composed of six exons with same length and five introns, indicating these genes were conserved and might originate from gene duplication during the evolution. Selection pressure analysis showed that MGgLYZ1 was under nearly neutral selection while MGgLYZ2 evolved under positive selection pressure with three positively selected amino acid residues (Y(102, L(200 and S(202 detected in the mature peptide. All these findings suggested MGgLYZ2 perhaps served as a digestive lysozyme under positive selection pressure during the evolution while MGgLYZ1 was mainly involved in innate immune responses.

  6. Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

    Directory of Open Access Journals (Sweden)

    F.C. Cançado


    Full Text Available cDNA coding for two digestive lysozymes (MdL1 and MdL2 of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8 at low ionic strength (μ = 0.02, which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (μ = 0.2. However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9 is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI indicate that MdL2 (pI = 6.7 is less positively charged than MdL1 (pI = 7.7 at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

  7. The Limitations of an Exclusively Colloidal View of Protein Solution Hydrodynamics and Rheology (United States)

    Sarangapani, Prasad S.; Hudson, Steven D.; Migler, Kalman B.; Pathak, Jai A.


    Proteins are complex macromolecules with dynamic conformations. They are charged like colloids, but unlike colloids, charge is heterogeneously distributed on their surfaces. Here we overturn entrenched doctrine that uncritically treats bovine serum albumin (BSA) as a colloidal hard sphere by elucidating the complex pH and surface hydration-dependence of solution viscosity. We measure the infinite shear viscosity of buffered BSA solutions in a parameter space chosen to tune competing long-range repulsions and short-range attractions (2 mg/mL ≤ [BSA] ≤ 500 mg/mL and 3.0 ≤ pH ≤ 7.4). We account for surface hydration through partial specific volume to define volume fraction and determine that the pH-dependent BSA intrinsic viscosity never equals the classical hard sphere result (2.5). We attempt to fit our data to the colloidal rheology models of Russel, Saville, and Schowalter (RSS) and Krieger-Dougherty (KD), which are each routinely and successfully applied to uniformly charged suspensions and to hard-sphere suspensions, respectively. We discover that the RSS model accurately describes our data at pH 3.0, 4.0, and 5.0, but fails at pH 6.0 and 7.4, due to steeply rising solution viscosity at high concentration. When we implement the KD model with the maximum packing volume fraction as the sole floating parameter while holding the intrinsic viscosity constant, we conclude that the model only succeeds at pH 6.0 and 7.4. These findings lead us to define a minimal framework for models of crowded protein solution viscosity wherein critical protein-specific attributes (namely, conformation, surface hydration, and surface charge distribution) are addressed. PMID:24268154

  8. Production and characterization of stable foams with fine bubbles from solutions of hydrophobin HFBII and its mixtures with other proteins

    NARCIS (Netherlands)

    Dimitrova, Lydia M.; Petkov, Plamen V.; Kralchevsky, Peter A.; Stoyanov, Simeon D.; Pelan, Eddie G.


    Hydrophobins are proteins that are excellent foam stabilizers. We investigated the effects of pH and addition of other proteins on the foaminess, bubble size, and stability of foams from aqueous solutions of the protein HFBII hydrophobin. The produced stable foams have bubbles of radii smaller than

  9. Investigation of a solvent-cast organogel to form a liquid-gel microinterface array for electrochemical detection of lysozyme. (United States)

    Felisilda, Bren Mark B; Alvarez de Eulate, Eva; Arrigan, Damien W M


    Ion transfer at aqueous-organogel interfaces enables the non-redox detection of ions and ionisable species by voltammetry. In this study, a non-thermal method for preparation of an organogel was employed and used for the detection of hen-egg-white-lysozyme (HEWL) via adsorptive stripping voltammetry at an array of aqueous-organogel microinterfaces. Tetrahydrofuran solvent casting was employed to prepare the organogel mixture, hence removing the need for heating of the solution to be gelled, as used in previous studies. Cyclic voltammetry of HEWL at the microinterface array revealed a broad adsorption process on the forward scan, at positive applied potentials, followed by a desorption peak at ca. 0.68 V, indicating the detection of HEWL in this region. Application of an adsorption step, where a constant optimized potential of 0.95 V was applied, followed by voltammetric detection provided for a linear response range of 0.02-0.84 μM and a detection limit of 0.030 μM for 300 s adsorption. The detection limit was further improved by utilizing differential pulse stripping voltammetry, resulting in detection limits of 0.017 μM, 0.014 μM, and 0.010 μM for adsorptive pre-concentration times of 60, 120 and 300 s, respectively, in unstirred solutions. These results are an improvement over other methods for the detection of HEWL at aqueous-organic interfaces and offers a basis for the label-free detection of protein.

  10. Structure and dynamics of bacteriophage IKe major coat protein in MPG micelles by solution NMR. (United States)

    Williams, K A; Farrow, N A; Deber, C M; Kay, L E


    The structure and dynamics of the 53-residue filamentous bacteriophage IKe major coat protein in fully protonated myristoyllysophosphatidylglycerol (MPG) micelles were characterized using multinuclear solution NMR spectroscopy. Detergent-solubilized coat protein [sequence: see text] mimics the membrane-bound "assembly intermediate" form of the coat protein which occurs during part of the phage life cycle. NMR studies of the IKe coat protein show that the coat protein is largely alpha-helical, exhibiting a long amphipathic surface. helix (Asn 4 to Ser 26) and a shorter "micelle-spanning" C-terminal helix which begins at TRP 29 and continues at least to Phe 48. Pro 30 likely occurs in the first turn of the C-terminal helix, where it is ideally situated given the hydrogen bonding and steric restrictions imposed by this residue. The similarity of 15N relaxation values (T1, T2, and NOE and 500 MHz and T2 at 600 MHz) among much of the N-terminal helix and all of the TM helix indicates that the N-terminal helix is as closely associated with the micelle as the TM helix. The description of the protein in the micelle is supported by the observation of NOEs between lysolipid protons and protein amide protons between asn 8 and Ser 50. The N-terminal and TM helices exhibit substantial mobility on the microsecond to second time scale, which likely reflects changes in the orientation between the two helices. The overall findings serve to clarify the role of individual residues in the context of a TM alpha-helix and provide an understanding of the secondary structure, dynamics, and aqueous and micellar environments of the coat protein.

  11. Complex coacervate core micelles with a lysozyme-modified corona

    NARCIS (Netherlands)

    Danial, M.; Klok, H.A.; Norde, W.; Cohen Stuart, M.A.


    This paper describes the preparation, characterization, and enzymatic activity of complex coacervate core micelles (C3Ms) composed of poly(acrylic acid) (PAA) and poly(N-methyl-2-vinyl pyridinium iodide)-b-poly(ethylene oxide) (PQ2VP-PEO) to which the antibacterial enzyme lysozyme is end-attached.

  12. 21 CFR 862.1490 - Lysozyme (muramidase) test system. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lysozyme (muramidase) test system. 862.1490 Section 862.1490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test...


    NARCIS (Netherlands)



    Lysozyme-induced fusion of phosphatidylserine (PS) vesicles was studied as a function of pH. Fusion, monitored by lipid-mixing, was measured by following the dilution of pyrene-labelled phosphatidylcholine, incorporated in PS vesicles, into unlabelled bilayers. It is demonstrated that

  14. Human serum albumin adsorption on TiO2 from single protein solutions and from plasma. (United States)

    Sousa, S R; Moradas-Ferreira, P; Saramago, B; Melo, L Viseu; Barbosa, M A


    In the present work, the adsorption of human serum albumin (HSA) on commercially pure titanium with a titanium oxide layer formed in a H(2)O(2) solution (TiO(2) cp) and on TiO(2) sputtered on Si (TiO(2) sp) was analyzed. Adsorption isotherms, kinetic studies, and work of adhesion determinations were carried out. HSA exchangeability was also evaluated. Surface characterization was performed by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and wettability studies. The two TiO(2) surfaces have very distinct roughnesses, the TiO(2) sp having a mean R(a) value 14 times smaller than the one of TiO(2) cp. XPS analysis revealed consistent peaks representative of TiO(2) on sputtered samples as well as on Ti cp substrate after 48 h of H(2)O(2) immersion. Nitrogen was observed as soon as protein was present, while sulfur, present in disulfide bonds in HSA, was observed for concentrations of protein higher than 0.30 mg/mL. The work of adhesion was determined from contact angle measurements. As expected from the surface free energy values, the work of adhesion of HSA solution is higher for the TiO(2) cp substrate, the more hydrophilic one, and lower for the TiO(2) sp substrate, the more hydrophobic one. The work of adhesion between plasma and the substrates assumed even higher values for the TiO(2) cp surface, indicating a greater interaction between the surface and the complex protein solutions. Adsorption studies by radiolabeling of albumin ((125)I-HSA) suggest that rapid HSA adsorption takes place on both surfaces, reaching a maximum value after approximately 60 min of incubation. For the higher HSA concentrations in solution, a multilayer coverage was observed on both substrates. After the adsorption step from single HSA solutions, the exchangeability of adsorbed HSA molecules by HSA in solution was evaluated. The HSA molecules adsorbed on TiO(2) sp seem to be more easily exchanged by HSA itself than those adsorbed on TiO(2) cp after 24 h. In

  15. Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides. (United States)

    Wei, Shina; Huang, Youhua; Cai, Jia; Huang, Xiaohong; Fu, Jing; Qin, Qiwei


    Lysozymes are key proteins of the host innate immune system against pathogen infection. In this study, a c-type lysozyme gene (Ec-lysC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysC cDNA is composed of 533 bp and encodes a polypeptide of 144-residue protein with 94% identity to lysC of Kelp grouper, Epinephelus bruneus. The genomic DNA of Ec-lysC consists of 4 exons and 3 introns, with a total length of 1897 bp. Amino acid sequence alignment showed that Ec-lysC possessed conserved catalytic residues (Glu50 and Asp67) and "GSTDYGIFQINS" motif. RT-PCR results showed that Ec-lysC transcript was most abundant in head kidney and less in muscle. The expression of Ec-lysC was differentially up-regulated in head kidney after stimulation with lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-lysC was distributed predominantly in the cytoplasm. The recombinant Ec-lysC (rEc-lysC) had lytic activities against Gram-positive bacteria Micrococcus lysodeikticus, Staphylococcus aureus, Streptococcus iniae and Gram-negative bacteria V. alginolyticus. The lysozyme acted on M. lysodeikticus cell walls as shown by scanning electron microscopy (SEM). Furthermore, overexpression of Ec-lysC in grouper cells delayed the occurrence of CPE induced by SGIV and inhibited the viral gene transcription significantly. Taken together, Ec-lysC might play an important role in grouper innate immune responses to invasion of bacterial and viral pathogens. C-type lysozyme gene from E. coioides (Ec-lysC) was identified and characterized.

  16. Crude protein level of pre-starter diets and nutritive solution for broilers

    Directory of Open Access Journals (Sweden)

    Rodrigo Santana Toledo


    Full Text Available The objective of the present study was to evaluate the effect of dietary crude protein (CP levels and the use of a nutritive solution via drinking water on the performance of pre-starter broilers. A total of 1,224 male Avian Farm chicks, from one to 40 days of age were used. Birds were distributed in a completely randomized experimental design with a 3 × 2 × 2 factorial arrangement, consisting of three initial body weights (low: 41 g, standard: 45 g and high: 49 g; two crude protein levels at the pre-starter phase (22 and 25% CP,with or without the addition of a nutritive solution, whose nutritional level was similar to the 25% CP pre-starter diet, at a concentration of 5% of the drinking water. Each treatment included six replicates and 17 chicks per experimental unit. At the end of the pre-starter phase, all hens received a diet with 22% CP until day 21 and a diet with 20% CP from the 21st to the 40th day. The use of the pre-starter diet with higher nutritional levels and the nutritive solution enhanced broiler performance. The early nutrient supply via drinking water resulted in better broiler performance and uniformity. However, birds with low initial body weight continued to present lower body weight at market age.

  17. Whey protein solution coating for fat-uptake reduction in deep-fried chicken breast strips. (United States)

    Dragich, Ann M; Krochta, John M


    This study investigated the use of whey protein, as an additional coating, in combination with basic, well-described predust, batter, and breading ingredients, for fat-uptake reduction in fried chicken. Chicken breasts were cut into strips (1 x 5 x 10 cm) and coated with wheat flour (WF) as a predust, dipped in batter, coated with WF as a breading, then dipped in 10% denatured whey protein isolate (DWPI) aqueous solution (wet basis). A WF-batter-WF treatment with no DWPI solution dip was included as a control. Coated chicken strips were deep-fried at 160 degrees C for 5 min. A Soxhlet-type extraction was performed to determine the fat content of the meat fraction of fried samples, the coating fraction of fried samples, raw chicken, and raw coating ingredients. The WF-batter-WF-10% DWPI solution had significantly lower fat uptake than the WF-batter-WF control, by 30.67% (dry basis). This article describes applied research involving fat reduction in coated deep-fried chicken. The methods used in this article were intended to achieve maximized fat reduction while maintaining a simple procedure applicable to actual food processing lines.

  18. Protein Crystallization (United States)

    Chernov, Alexander A.


    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  19. Enhancement and suppression of protein crystal nucleation due to electrically driven convection (United States)

    Penkova, Anita; Gliko, Olga; Dimitrov, Ivaylo L.; Hodjaoglu, Feyzim V.; Nanev, Christo; Vekilov, Peter G.


    We investigated the effects of the constant electric fields from 2.0 to 6.0 kV cm -1 on the nucleation of ferritin, apoferritin and lysozyme crystals. For this, supersaturated solutions of the three proteins were held between electrodes separated by 1.0 cm in batch and sitting drop geometries without contact between electrodes and solutions. The nucleation rate was characterized by the number of crystals appearing after a certain time (1-3 days). We show that in sitting drop arrangements, weak electric fields (<4 kV cm -1) either suppress or have no effect on the nucleation rate of ferritin and apoferritin, while electric fields of 5 or 6 kV cm -1 reproducibly enhance crystal nucleation of both proteins. Electric fields of all tested strengths consistently enhance lysozyme crystal nucleation. All batch experiments showed no effect of the electric field on the nucleation rates. Since the solutions contain high electrolyte concentrations and are conductive, the electric field strengths within them are negligible. We show that the electric field causes solution stirring with rates of up to 100 μm s -1, depending of the field strength. Thus, our observations indicate that at slow solution flow rates, the rates of nucleation of ferritin and apoferritin crystal are suppressed, while faster stirring enhances crystal nucleation of these proteins. All solution flow rates enhance lysozyme crystal nucleation. Our results suggest that solution convection may strongly affect nucleation, and that for some systems, an optimal convection velocity, leading to fastest nucleation, exists.

  20. Evaluation of activity of selected antioxidants on proteins in solution and in emulsions. (United States)

    Baron, Caroline P; Berner, Lis; Skibsted, Leif H; Refsgaard, Hanne H F


    Protection against protein oxidation by lipophilic and hydrophilic antioxidants in model systems using bovine serum albumin (BSA) in solution alone, or in an emulsion with linolenic acid methyl ester (LnMe) was found to be strongly dependent on the oxidation initiator. Tocopherol, Trolox, or the carotenoids astaxanthin and canthaxanthin were incubated with BSA or BSA/LnMe and oxidation was initiated either with the water-soluble azo-initiator 2,2' azo-bis-(2-amidinopropane) hydrochloride (AAPH), or FeCl3 and ascorbate, or the Fenton system using FeCl2/EDTA/H2O2, or with the singlet oxygen generating species anthracene-9,10-dipropionic acid disodium 1,4 endoperoxide (NDPO2). The results show that all the antioxidants tested were inefficient in the system with FeCl3/ascorbate. However, with the other initiating agents, the hydrophilic antioxidant, Trolox, was the most effective in preventing both protein and lipid oxidation. In contrast the lipophilic antioxidants were ineffective in preventing oxidation of BSA in aqueous solution, but did show some moderate antioxidative activity on protein and lipid in the BSA/LnMe system. Using the singlet oxygen generating system it was also demonstrated that Trolox always provided better protection of the protein than tocopherol and the carotenoids in both the BSA and the BSA/LnMe systems. In conclusion, prevention of protein oxidation using a water-soluble antioxidant has a protective effect on the lipid fraction and this approach deserves further attention in complex biological systems.

  1. Use of Lysozyme as a Feed Additive on Rumen Fermentation and Methane Emission

    Directory of Open Access Journals (Sweden)

    Ashraf A. Biswas


    Full Text Available This study was conducted to determine the effect of lysozyme addition on in vitro rumen fermentation and to identify the lysozyme inclusion rate for abating methane (CH4 production. An in vitro ruminal fermentation technique was done using a commercial concentrate to rice straw ratio of 8:2 as substrate. The following treatments were applied wherein lysozyme was added into 1 mg dry matter substrate at different levels of inclusion: Without lysozyme, 2,000, 4,000, and 8,000 U lysozyme. Results revealed that, lysozyme addition had a significant effect on pH after 24 h of incubation, with the highest pH (p<0.01 observed in 8,000 U lysozyme, followed by the 4,000 U, 2,000 U, and without lysozyme. The highest amounts of acetic acid, propionic acid (p<0.01 and total volatile fatty acid (TVFA (p<0.05 were found in 8,000 U after 24 h of incubation. The CH4 concentration was the lowest in the 8,000 U and the highest in the without lysozyme addition after 24 h of incubation. There was no significant differences in general bacteria, methanogen, or protozoan DNA copy number. So far, addition of lysozyme increased the acetate, propionate, TVFA, and decreased CH4 concentration. These results suggest that lysozyme supplementation may improve in vitro rumen fermentation and reduce CH4 emission.

  2. Effective approach to greatly enhancing selective secretion and expression of three cytoplasmic enzymes in Escherichia coli through synergistic effect of EDTA and lysozyme. (United States)

    Liu, Sen-Lin; Du, Kun; Chen, Wei-Zhao; Liu, Gang; Xing, Miao


    An effective approach to greatly enhancing the selective secretion and expression of recombinant cytoplasmic enzymes in Escherichia coli was successfully developed through the synergistic effect of ethylenediaminetetraacetate (EDTA) and lysozyme. The method was applied to two endoglucanases (EGs) and an amylase. The optimal culture conditions of temperature and concentration of isopropyl-β-D: -1-thiogalactopyranoside (IPTG) were 23-30 °C and 0.2 mM, respectively, under which the three enzymes could be expressed in active form. Among all the chemicals tested, EDTA was found to be most suitable for enhancing the secretion of EG-I-1A into the medium. Addition of lysozyme alone had little influence on the secretion and expression. In contrast, on the basis of the addition of 5 g EDTA/L at the induction time of 12 h, the simultaneous addition of 0.15 g lysozyme/L further significantly increased the secretion and expression of the three enzymes, demonstrating the synergistic effect of EDTA and lysozyme. The production of EG-I-1A in the culture medium by adding 5 g EDTA/L and 0.15 g lysozyme/L under the optimal culture conditions of 23 °C and 0.2 mM IPTG was over 260-fold higher than that without EDTA and lysozyme under the standard conditions of 37 °C and 1 mM IPTG. In summary, the advantage of this novel cultivation approach for secretion was that not only did it selectively enhance the secretion of the proteins of interest, but also greatly increased the expression of the three enzymes by over 80 %.

  3. Solution structure of the equine infectious anemia virus p9 protein: a rationalization of its different ALIX binding requirements compared to the analogous HIV-p6 protein

    National Research Council Canada - National Science Library

    Sharma, Alok; Bruns, Karsten; Röder, René; Henklein, Peter; Votteler, Jörg; Wray, Victor; Schubert, Ulrich


    ... structure and folding of this 51-amino acid protein under different solution conditions. Qualitative 1H-chemical shift and NOE data indicate that in a pure aqueous environment p9 favors an unstructured state...

  4. Chicken-type lysozyme in channel catfish: Expression analysis, lysozyme activity and efficacy as immunostimulant against Aeromonas hydrophila infection (United States)

    To understand whether chicken-type lysozyme (Lys-c) in channel catfish was induced by infection of Aeromonas hydrophila, the transcriptional levels of Lys-c in skin, gut, liver, spleen, posterior kidney, and blood cells in healthy channel catfish was compared to that in channel catfish infected with...


    Directory of Open Access Journals (Sweden)

    Maria de Hoyos Guajardo, Ph.D. Candidate, M.Sc., B.Eng.


    Full Text Available The theory that is presented below aims to conceptualise how a group of undergraduate students tackle non-routine mathematical problems during a problem-solving course. The aim of the course is to allow students to experience mathematics as a creative process and to reflect on their own experience. During the course, students are required to produce a written ‘rubric’ of their work, i.e., to document their thoughts as they occur as well as their emotionsduring the process. These ‘rubrics’ were used as the main source of data.Students’ problem-solving processes can be explained as a three-stage process that has been called ‘solutioning’. This process is presented in the six sections below. The first three refer to a common area of concern that can be called‘generating knowledge’. In this way, generating knowledge also includes issues related to ‘key ideas’ and ‘gaining understanding’. The third and the fourth sections refer to ‘generating’ and ‘validating a solution’, respectively. Finally, once solutions are generated and validated, students usually try to improve them further before presenting them as final results. Thus, the last section deals with‘improving a solution’. Although not all students go through all of the stages, it may be said that ‘solutioning’ considers students’ main concerns as they tackle non-routine mathematical problems.

  6. Solution structure of FK506-binding protein 12 from Aedes aegypti. (United States)

    Chakraborty, Goutam; Shin, Joon; Nguyen, Quoc Toan; Harikishore, Amaravadhi; Baek, Kwanghee; Yoon, Ho Sup


    Dengue remains one of the major public concerns as the virus eludes the immune response. Currently, no vaccines or antiviral therapeutics are available for dengue prevention or treatment. Immunosuppressive drug FK506 shows an antimalarial activity, and its molecular target, FK506-binding protein (FKBP), was identified in human Plasmodium parasites. Likewise, a conserved FKBP family protein has also been identified in Aedes aegypti (AaFKBP12), which is expected to play a similar role in the life cycle of Aedes aegypti, the primary vector of dengue virus infection. As FKBPs belong to a highly conserved class of immunophilin family and are involved in key biological regulations, they are considered as attractive pharmacological targets. In this study, we have determined the nuclear magnetic resonance solution structure of AaFKBP12, a novel FKBP member from Aedes aegypti, and presented its structural features, which may facilitate the design of potential inhibitory ligands against the dengue-transmitting mosquitoes.

  7. Proteins in frozen solutions: evidence of ice-induced partial unfolding. (United States)

    Strambini, G B; Gabellieri, E


    From a drastic decrease in the phosphorescence lifetime of tryptophan residues buried in compact rigid cores of globular proteins, it was possible to demonstrate that freezing of aqueous solutions is invariably accompanied by a marked loosening of the native fold, an alteration that entails considerable loss of secondary and tertiary structure. The phenomenon is largely reversible on ice melting although, in some cases, a small fraction of macromolecules recovers neither the initial phosphorescence properties nor the catalytic activity. The variation in the lifetime parameter was found to be a smooth function of the residual volume of liquid water in equilibrium with ice and to depend on the morphology of ice. The addition of cryoprotectants such as glycerol and sucrose profoundly attenuates or even eliminates the perturbation. These results are interpreted in terms of adsorption of protein molecules onto the surface of ice.

  8. Microscale thermophoresis as a sensitive method to quantify protein: nucleic acid interactions in solution. (United States)

    Zillner, Karina; Jerabek-Willemsen, Moran; Duhr, Stefan; Braun, Dieter; Längst, Gernot; Baaske, Philipp


    Microscale thermophoresis (MST) is a new method that enables the quantitative analysis of molecular interactions in solution at the microliter scale. The technique is based on the thermophoresis of molecules, which provides information about molecule size, charge, and hydration shell. Since at least one of these parameters is typically affected upon binding, the method can be used for the analysis of each kind of biomolecular interaction or modification of proteins or DNA. Quantitative binding parameters are obtained by using a serial dilution of the binding substrate. This section provides a detailed protocol describing the analysis of DNA-protein interactions, using the AT-hook peptides as a model system that bind to short double-stranded DNA.


    Directory of Open Access Journals (Sweden)

    Maura P. Alves


    Full Text Available This study aimed to determine viscosity curves prepared from whey protein concentrates (WPCs by the rapid viscosity analyzer (RVA and determine the optimal heat treatment time in order to obtain the maximum viscosity solutions at this stage. The WPCs produced from whey samples initially subjected to thermal treatment and microfiltration presented composition compatible with the international standards, with a significant difference (p<0.05 for fat concentration. Viscographic profiles indicated that WPCs produced from microfiltered whey had higher viscosities than those subjected to heat treatment. In addition, 10 min was determined to be the optimal length of time for heat treatment in order to maximise WPCs viscosity. These results indicate that WPC production can be designed for different food applications. Finally, a rapid viscosity analyzer was demonstrated to be an appropriate tool to study the application of whey proteins in food systems.

  10. Solution NMR Studies of Mycobacterium tuberculosis Proteins for Antibiotic Target Discovery

    Directory of Open Access Journals (Sweden)

    Do-Hee Kim


    Full Text Available Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis, which triggers severe pulmonary diseases. Recently, multidrug/extensively drug-resistant tuberculosis strains have emerged and continue to threaten global health. Because of the development of drug-resistant tuberculosis, there is an urgent need for novel antibiotics to treat these drug-resistant bacteria. In light of the clinical importance of M. tuberculosis, 2067 structures of M. tuberculsosis proteins have been determined. Among them, 52 structures have been solved and studied using solution nuclear magnetic resonance (NMR. The functional details based on structural analysis of M. tuberculosis using NMR can provide essential biochemical data for the development of novel antibiotic drugs. In this review, we introduce diverse structural and biochemical studies on M. tuberculosis proteins determined using NMR spectroscopy.

  11. Influences of urea, pH and metal ions on the interaction between cepharanthine and lysozyme by steady state fluorescence spectroscopy (United States)

    Yang, Yumin; Li, Daojin; Xu, Chen


    The study on the binding mode of drug with protein is important to understand the pharmacokinetics and toxicity of the drug as well as the relationship of structure and function of the protein. In the study, the interaction between cepharanthine and lysozyme (Lys) in aqueous solution was first investigated by fluorescence spectroscopic techniques at pH 7.4. The obtained quenching rate constant and binding constant indicated the static quenching mechanism and medium binding force. The effect of cepharanthine on the conformation of Lys was analyzed using synchronous fluorescence and three-dimensional (3D) fluorescence. In addition, the effect of urea on the interaction of cepharanthine with Lys was studied and the binding capacity of cepharanthine to the denatured Lys deceases dramatically, as compared with that of cepharanthine to native Lys. Moreover, influence of pH on the interaction of cepharanthine with Lys was investigated. As compared with that at pH 7.4, the binding abilities of the drug to Lys under other pH conditions (pH 9.0, 5.5, 3.5, and 1.9) deceased. Furthermore, the effect of metal ions on the binding constant of cepharanthine with Lys was investigated.

  12. Primary structure and solution conditions determine conformational ensemble properties of intrinsically disordered proteins (United States)

    Mao, Hsuan-Han Alberto

    Intrinsically disordered proteins (IDPs) are a class of proteins that do not exhibit well-defined three-dimensional structures. The absence of structure is intrinsic to their amino acid sequences, which are characterized by low hydrophobicity and high net charge per residue compared to folded proteins. Contradicting the classic structure-function paradigm, IDPs are capable of interacting with high specificity and affinity, often acquiring order in complex with protein and nucleic acid binding partners. This phenomenon is evident during cellular activ