Stability Of Proteins In Solution Research Articles

DNA-based assay for calorimetric determination of protein concentrations in pure or mixed solutions.

It was recently reported that values of the transition heat capacities, as measured by differential scanning calorimetry, for two globular proteins and a short DNA hairpin in NaCl buffer are essentially equivalent, at equal concentrations (mg/mL). To validate the broad applicability of this phenomenon, additional evidence for this equivalence is presented that reveals it does not depend on DNA sequence, buffer salt, or transition temperature (Tm). Based on the equivalence of transition heat capacities, a calorimetric method was devised to determine protein concentrations in pure and complex solutions. The scheme uses direct comparisons between the thermodynamic stability of a short DNA hairpin standard of known concentration, and thermodynamic stability of protein solutions of unknown concentrations. Sequences of two DNA hairpins were designed to confer a near 20°C difference in their Tm values. In all cases, evaluated protein concentrations determined from the DNA standard curves agreed with the UV-Vis concentration for monomeric proteins. For multimeric proteins evaluated concentrations were greater than determined by UV-Vis suggesting the calorimetric approach can also be an indicator of molecular stoichiometry.

Predicting Colloidal Stability of High-Concentration Monoclonal Antibody Formulations in Common Pharmaceutical Buffers Using Improved Polyethylene Glycol Induced Protein Precipitation Assay.

Colloidal stability is an important consideration when developing high concentration mAb formulations. PEG-induced protein precipitation is a commonly used assay to assess the colloidal stability of protein solutions. However, the practical usefulness and the current theoretical model for this assay have yet to be verified over a large formulation space across multiple mAbs and mAb-based modalities. In the present study, we used PEG-induced protein precipitation assays to evaluate colloidal stability of 3 mAbs in 24 common formulation buffers at 20 and 5 °C. These prediction assays were conducted at low protein concentration (1 mg/mL). We also directly characterized high concentration (100 mg/mL) formulations for cold-induced phase separation, turbidity, and concentratibility by ultrafiltration. This systematic study allowed analysis of the correlation between the results of low concentration assays and the high concentration attributes. The key findings of this study include the following: (1) verification of the usefulness of three different parameters (Cmid, μB, and Tcloud) from PEG-induced protein precipitation assays for ranking colloidal stability of high concentration mAb formulations; (2) a new method to implement PEG-induced protein precipitation assay suitable for high throughput screening with low sample consumption; (3) improvement in the theoretical model for calculating robust thermodynamic parameters of colloidal stability (μB and εB) that are independent of specific experimental settings; (4) systematic evaluation of the effects of pH and buffer salts on colloidal stability of mAbs in common formulation buffers. These findings provide improved theoretical and practical tools for assessing the colloidal stability of mAbs and mAb-based modalities during formulation development.

Effects of Hydrogen/Deuterium Exchange on Protein Stability in Solution and in the Gas Phase.

Mass spectrometry (MS)-based techniques are widely used for probing protein structure and dynamics in solution. H/D exchange (HDX)-MS is one of the most common approaches in this context. HDX is often considered to be a "benign" labeling method, in that it does not perturb protein behavior in solution. However, several studies have reported that D2O pushes unfolding equilibria toward the native state. The origin, and even the existence of this protein stabilization remain controversial. Here we conducted thermal unfolding assays in solution to confirm that deuterated proteins in D2O are more stable, with 2-4 K higher melting temperatures than unlabeled proteins in H2O. Previous studies tentatively attributed this phenomenon to strengthened H-bonds after deuteration, an effect that may arise from the lower zero-point vibrational energy of the deuterated species. Specifically, it was proposed that strengthened water-water bonds (W···W) in D2O lower the solubility of nonpolar side chains. The current work takes a broader view by noting that protein stability in solution also depends on water-protein (W···P) and protein-protein (P···P) H-bonds. To help unravel these contributions, we performed collision-induced unfolding (CIU) experiments on gaseous proteins generated by native electrospray ionization. CIU profiles of deuterated and unlabeled proteins were indistinguishable, implying that P···P contacts are insensitive to deuteration. Thus, protein stabilization in D2O is attributable to solvent effects, rather than alterations of intraprotein H-bonds. Strengthening of W···W contacts represents one possible explanation, but the stabilizing effect of D2O can also originate from weakened W···P bonds. Future work will be required to elucidate which of these two scenarios is correct, or if both contribute to protein stabilization in D2O. In any case, the often-repeated adage that "D-bonds are more stable than H-bonds" does not apply to intramolecular contacts in native proteins.

Phosphorylated resveratrol as a protein aggregation suppressor in vitro and in vivo.

The stability of proteins in solution poses a great challenge for both technical applications and molecular biology, including neurodegenerative diseases. In this work, a phosphorylated resveratrol material was examined for its anti-aggregation properties in vitro and in vivo. Here, an anti-fibrillation effect could be measured for amyloid beta and human insulin in vitro and general anti-aggregation properties for crude chicken egg white in solution. Using a drosophila fly model for the overexpression of amyloid beta protein, changes in physiological protein aggregation and improved locomotor abilities could be observed in the presence of dietary phosphorylated resveratrol.

Synthesis and Characterization of Cholesteryl Conjugated Lysozyme (CHLysozyme).

Hydrophobic interaction is important for protein conformation. Conjugation of a hydrophobic group can introduce intermolecular hydrophobic contacts that can be contained within the molecule. It is possible that a strongly folded state can be formed in solution compared with the native state. In this study, we synthesized cholesteryl conjugated lysozyme (CHLysozyme) using lysozyme and cholesterol as the model protein and hydrophobic group, respectively. Cholesteryl conjugation to lysozyme was confirmed by nuclear-magnetic resonance. Differential-scanning calorimetry suggested that CHLysozyme was folded in solution. CHLysozyme secondary structure was similar to lysozyme, although circular dichroism spectra indicated differences to the tertiary structure. Fluorescence measurements revealed a significant increase in the hydrophobic surface of CHLysozyme compared with that of lysozyme; CHLysozyme self-associated by hydrophobic interaction of the conjugated cholesterol but the hydrophobic surface of CHLysozyme decreased with time. The results suggested that hydrophobic interaction changed from intramolecular interaction to an intermolecular interaction. Furthermore, the relative activity of CHLysozyme to lysozyme increased with time. Therefore, CHLysozyme likely forms a folded state with an extended durability of activity. Moreover, lysozyme was denatured in 100% DMSO but the local environment of tryptophan in CHLysozyme was similar to that of a native lysozyme. Thus, this study suggests that protein solution stability and resistance to organic solvents may be improved by conjugation of a hydrophobic group.

Conformational Stability and Dynamics in Crystals Recapitulate Protein Behavior in Solution

A growing body of evidences has established that in many cases proteins may preserve most of their function and flexibility in a crystalline environment, and several techniques are today capable to characterize molecular properties of proteins in tightly packed lattices. Intriguingly, in the case of amyloidogenic precursors, the presence of transiently populated states (hidden to conventional crystallographic studies) can be correlated to the pathological fate of the native fold; the low fold stability of the native state is a hallmark of aggregation propensity. It remains unclear, however, to which extent biophysical properties of proteins such as the presence of transient conformations or protein stability characterized in crystallo reflect the protein behavior that is more commonly studied in solution. Here, we address this question by investigating some biophysical properties of a prototypical amyloidogenic system, β2-microglobulin in solution and in microcrystalline state. By combining NMR chemical shifts with molecular dynamics simulations, we confirmed that conformational dynamics of β2-microglobulin native state in the crystal lattice is in keeping with what observed in solution. A comparative study of protein stability in solution and in crystallo is then carried out, monitoring the change in protein secondary structure at increasing temperature by Fourier transform infrared spectroscopy. The increased structural order of the crystalline state contributes to provide better resolved spectral components compared to those collected in solution and crucially, the crystalline samples display thermal stabilities in good agreement with the trend observed in solution. Overall, this work shows that protein stability and occurrence of pathological hidden states in crystals parallel their solution counterpart, confirming the interest of crystals as a platform for the biophysical characterization of processes such as unfolding and aggregation.

Probing the Effects of Heterogeneous Oxidative Modifications on the Stability of Cytochrome c in Solution and in the Gas Phase.

Covalent modifications by reactive oxygen species can modulate the function and stability of proteins. Thermal unfolding experiments in solution are a standard tool for probing oxidation-induced stability changes. Complementary to such solution investigations, the stability of electrosprayed protein ions can be assessed in the gas phase by collision-induced unfolding (CIU) and ion-mobility spectrometry. A question that remains to be explored is whether oxidation-induced stability alterations in solution are mirrored by the CIU behavior of gaseous protein ions. Here, we address this question using chloramine-T-oxidized cytochrome c (CT-cyt c) as a model system. CT-cyt c comprises various proteoforms that have undergone MetO formation (+16 Da) and Lys carbonylation (LysCH2-NH2 → LysCHO, -1 Da). We found that CT-cyt c in solution was destabilized, with a ∼5 °C reduced melting temperature compared to unmodified controls. Surprisingly, CIU experiments revealed the opposite trend, i.e., a stabilization of CT-cyt c in the gas phase. To pinpoint the source of this effect, we performed proteoform-resolved CIU on CT-cyt c fractions that had been separated by cation exchange chromatography. In this way, it was possible to identify MetO formation at residue 80 as the key modification responsible for stabilization in the gas phase. Possibly, this effect is caused by newly formed contacts of the sulfoxide with aromatic residues in the protein core. Overall, our results demonstrate that oxidative modifications can affect protein stability in solution and in the gas phase very differently.

Twin-arginine signal peptide of Bacillus licheniformis GlmU efficiently mediated secretory expression of protein glutaminase

CITATION: Niu, D., et al. 2019. Twin-arginine signal peptide of Bacillus licheniformis GlmU efficiently mediated secretory expression of protein glutaminase. Electronic Journal of Biotechnology, 42:49-55. doi:10.1016/j.ejbt.2019.10.006

Irreversible Nature of Mesoscopic Aggregates in Lysozyme Solutions

Study of protein stability in solutions is important for better understanding the pathogenesis of diseases caused by abnormal protein folding and aggregation. Lysozyme is known to form mesoscopic aggregates (30–100 nm radius) in concentrated solutions (>30 mg/mL), however the origin and thermodynamic status of these aggregates remain unclear. In this work we have investigated the effects of concentration, filtration, and temperature on the sizes and relative amount of mesoscale aggregates in solutions of lysozyme. We have used dynamic light scattering, small-angle X-ray scattering, and size exclusion chromatography (SEC). Mesoscopic protein aggregates were commonly thought to be in equilibrium with protein monomers in solution, resulting from a reversible self-assembly of the monomers. We instead show that systematic filtration through 20 nm pore size filters completely removes the aggregates from solution. The aggregates do not reemerge. Without filtering, the relative number of monomers decreases with increasing solution temperature, indicating formation of more aggregates. SEC was used to search for the presence of lysozyme dimers, which have been previously hypothesized to be related to the formation of mesoscopic aggregates. SEC did not detect dimers in solutions of filtered or unfiltered lysozyme. Taken together, our results strongly suggest that the mesoscopic aggregates in lysozyme are not caused by reversible self-assembly of lysozyme monomers and are not an intrinsic property of lysozyme monomers in their native state. We hypothesize that the lysozyme aggregation is likely due to some impurities in lysozyme introduced during purification or lyophilization and/or to traces of misfolded lysozyme.

Solution thermodynamic approach to analyze protein stability in aqueous solutions

Protein thermal stability was analyzed by a solution thermodynamic approach. The small energetic differences in hydrogen-bonds (HB) among amino acid resdues and water molecules were proved to be amplified by the large number of HB involved to bring about the equilibrium shift from folding to unfolding of proteins. In aqueous solutions, water activity (Aw) plays a key role in protein stability. Therefore, Aw was precisely determined for various solutions and its relationship with solution structure was discussed. Wyman-Tanford analysis based on Aw showed linear regressions, without exception, between protein unfolding-ratio and Aw for lysozyme, ribonuclease A, and α-chymotrypsinogen A in various solutions with sugars, osmolytes, alcohols, and protein denaturant. From this linear regression, the free energy difference, ΔΔG, for a protein in a solution and in pure water, was easily obtained. Protein stability in a solution was proved to be determined by a balance between hydration and solute-binding effects to the protein and also by solution structure, which indirectly affects the hydrophobic interaction in a protein molecule. Temperature dependence of HB on protein stability suggested its interrelationship with hydrophobic interaction.

Prediction and characterization of the stability enhancing effect of the Cherry-Tag™ in highly concentrated protein solutions by complex rheological measurements and MD simulations

Solution stability attributes are one of the key parameters within the production and launching phase of new biopharmaceuticals. Instabilities of active biological compounds can reduce the yield of biopharmaceutical productions, and may induce undesired reactions in patients, such as immunogenic rejections. Protein solution stability thus needs to be engineered and monitored throughout production and storage. In contrast to the gold standard of long-term storage experiments applied in industry, novel experimental and in silico molecular dynamics tools for predicting protein solution stability can be applied within several minutes or hours. Here, a rheological approach in combination with molecular dynamics simulations are presented, for determining and predicting long-term phase behavior of highly concentrated protein solutions. A diversity of liquid phase conditions, including salt type, ionic strength, pH and protein concentration are tested in a Glutathione-S-Transferase (GST) case study, in combination with the enzyme with and without solubility-enhancing Cherry-Tag™. The rheological characterization of GST and Cherry-GST solutions enabled a fast and efficient prediction of protein instabilities without the need of long-term protein phase diagrams. Finally, the strong solubility enhancing properties of the Cherry-Tag™ were revealed by investigating protein surface properties in MD simulations. The tag highly altered the overall surface charge and hydrophobicity of GST, making it less accessible to alteration by the chemical surrounding.

Sensitive Detection of Immunoglobulin G Stability Using in Real-Time Isothermal Differential Scanning Fluorimetry: Determinants of Protein Stability for Antibody-Based Therapeutics

Protein instability is a major obstacle in the production and delivery of monoclonal antibody–based therapies for cancer. This study presents real-time isothermal differential scanning fluorimetry as an emerging method to evaluate the stability of human immunoglobulin G protein with high sensitivity. The stability of polyclonal human immunoglobulin G against urea-induced denaturation was assessed following: (1) oxidation by the free-radical generator 2,2-Azobis[2-amidinopropane]dihydrochloride and (2) in selected storage buffers. Significant differences in immunoglobulin G stability were detected by real-time isothermal differential scanning fluorimetry when the immunoglobulin G was stored in 1,4-Piperazinediethanesulfonic acid buffer compared to phosphate-buffered saline, with half-maximal rate of denaturation occurring at a higher urea concentration in 1,4-Piperazinediethanesulfonic acid than phosphate-buffered saline (K nd;PIPES = 3.56 ± 0.09 M, K nd;PBS = 2.94 ± 0.08 M; P < .01), but differential scanning fluorimetry did not detect differences in unfolding temperature (T m;PIPES = 70.5 ± 0.3°C, T m;PBS = 69.7 ± 0.2°C). The effects of 2,2-Azobis[2-amidinopropane]dihydrochloride-induced oxidation on immunoglobulin G stability were analyzed by real-time isothermal differential scanning fluorimetry; the oxidized protein showed greater sensitivity to urea (K nd;CNTRL = 3.96 ± 0.19 M, K nd;AAPH = 3.49 ± 0.07 M; P < .05). Similarly, differential scanning fluorimetry indicated greater thermal sensitivity of oxidized immunoglobulin G (T m;CNTRL = 70.5 ± 0.3°C, T m;AAPH = 62.9 ± 0.1°C; P < .001). However, a third method for assessing protein stability, pulse proteolysis, proved to be substantially less sensitive and did not detect significant effects of 2,2-Azobis[2-amidinopropane]dihydrochloride on the half-maximal concentration of urea needed to denature immunoglobulin G (C m;CNTRL= 6.8 ± 0.1 M; C m;AAPH = 6.4 ± 0.7 M). Overall these results demonstrate the merit of using real-time isothermal differential scanning fluorimetry as a rapid and sensitive technique for the evaluation of protein stability in solution using a quantitative real-time thermocycler.

Bilateral Effects of Excipients on Protein Stability: Preferential Interaction Type of Excipient and Surface Aromatic Hydrophobicity of Protein.

Understanding the mechanism of protein-excipient interaction and illuminating the influencing factors on protein stability are key steps in the rational design of protein formulations. The objective of this study was to assess effects of preferential interaction type of excipient and surface aromatic hydrophobicity of protein on protein solution stability. The preferential interaction between excipient and aromatic hydrophobic area of protein was investigated by solubility and fluorescence studies of amino acid derivatives in excipient solutions. We examined conformational, colloidal and mechanical stabilities of model proteins with different surface aromatic hydrophobicities, including bovine serum albumin (BSA) and ovalbumin (OVA), and then stability data were visualized by three-index empirical phase diagram. The result showed that preferentially excluded excipients (trehalose, sucrose and sorbitol) protected protein conformation against damage, but they could accelerate mechanical stress-induced aggregation. Preferentially bound excipients (propanediol and arginine) suppressed BSA aggregation, but arginine failed to inhibit OVA aggregation, which might be attributed to the disparate conformational perturbing effects of arginine on aromatic hydrophobic regions of BSA and OVA. These findings provided strong evidence that excipient possessed bilateral effects, and its application should be determined on different preferential interaction behaviors of excipients with protein, especially with the aromatic hydrophobic region.

Correlating the Effects of Antimicrobial Preservatives on Conformational Stability, Aggregation Propensity, and Backbone Flexibility of an IgG1 mAb

Multidose formulations of biotherapeutics, which offer better dosage management and reduced production costs, require the addition of antimicrobial preservatives (APs). APs have been shown, however, to decrease protein stability in solution and cause protein aggregation. In this report, the effect of 4 APs, m-cresol, phenol, phenoxyethanol, and benzyl alcohol on conformational stability, aggregation propensity, and backbone flexibility of an IgG1 mAb, mAb-4, is investigated. Compared with no preservative control, each of the APs decreased the conformational stability of mAb-4 as measured by differential scanning calorimetry and extrinsic fluorescence spectroscopy. The addition of APs resulted in increased monomer loss and aggregate accumulation at 50°C over 28 days, as monitored by size-exclusion chromatography. The extent of conformational destabilization and protein aggregation of mAb-4 induced by APs followed their calculated octanol–water partition coefficients. Increases in backbone flexibility, as measured by hydrogen exchange, of a region located in the CH2 domain of the mAb (heavy chain 237-254) in the presence of APs also correlated with hydrophobicity. Based on these results, the destabilizing effect of APs on mAb-4 correlates with the increased hydrophobicity of the APs and their ability to enhance the local backbone flexibility of an aggregation hot spot within the CH2 domain of the mAb.

A combined approach for enhancing the stability of recombinant cis-dihydrodiol naphthalene dehydrogenase from Pseudomonas putida G7 allowed for the structural and kinetic characterization of the enzyme

The second enzyme of the naphthalene degradation pathway in Pseudomonas putida G7 is NahB, a dehydrogenase that converts cis-1,2-dihydroxy-1,2-dihydronaphthalene to 1,2-dihydroxynaphthalene. We report the cloning, optimization of expression, purification, kinetic studies and preliminary structural characterization of the recombinant NahB. The nahB gene was cloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli Rosetta (DE3) as an N-terminal hexa-histidine-tagged protein (6xHis-NahB). Using methods of enhancing protein stability in solution, we tested different expression, cell lysis, and purification protocols with and without ligand supplementation. The protein stability was evaluated by dynamic light scattering and circular dichroism spectroscopy assays. Best-derived protocols (expression at 18 °C, cell lysis with homogenizer, and three purification steps) were used to produce 20 mg of homogeneous 6xHis-NahB per liter of culture. The secondary and quaternary structures of 6xHis-NahB were assessed by circular dichroism and size-exclusion chromatography experiments, respectively. The enzyme was NAD+-dependent and active at pH 7.0 and 9.4 for the oxidation of the substrate. The Michaelis-Menten parameters determined at pH 7.0 and 25 °C for the substrate and cofactor, presented respective Km values of 6 and 350 μM, and a kcat value of 8.3 s−1. Furthermore, we identified conditions for the crystallization of 6xHis-NahB. X-ray diffraction data were collected from a single 6xHis-NahB crystal which diffracted to 2.21 Å. The crystal belongs to space group I222, with unit-cell parameters a = 63.62, b = 69.50, and c = 117.47 Å. The tertiary structure of 6xHis-NahB was determined using the molecular replacement method. Further structural refinement is currently underway.

The effect of deuterium oxide on the conformational stability and aggregation of bovine serum albumin

Protein aggregation is a significant problem affecting the integrity of proteins, and is a major hindrance to the development of biopharmaceutical products. Deuterium oxide (D2O), widely used in protein characterization studies, has been shown to promote protein aggregation when used as a substitute for water in most buffered protein solutions; however, a few studies have reported minor improvements in melting point temperatures for some proteins. Our study aims to investigate the effect of D2O on protein stability, using bovine serum albumin (BSA) as a model. We performed accelerated stability studies at high temperatures and assessed the physical and conformational stability of BSA using fluorescence spectroscopy, dynamic light scattering (DLS) and size-exclusion high performance liquid chromatography. Our findings reveal that D2O enhances the conformational stability of monomeric BSA, reducing monomer loss and formation of small aggregates at high temperatures. There is also an increase in the formation of larger aggregates probed by thioflavin T (ThT), however, the increase is not considered significant based on DLS results. Our findings demonstrate that exchanging water with D2O can improve the stability of proteins in solution, by maintaining the stability of the monomeric form, which may be beneficial for the long-term storage of some biological products.

Evaluation of effects of pH and ionic strength on colloidal stability of IgG solutions by PEG-induced liquid-liquid phase separation.

Colloidal stability of IgG antibody solutions is important for pharmaceutical and medicinal applications. Solution pH and ionic strength are two key factors that affect the colloidal stability of protein solutions. In this work, we use a method based on the PEG-induced liquid-liquid phase separation to examine the effects of pH and ionic strength on the colloidal stability of IgG solutions. We found that at high ionic strength (≥0.25M), the colloidal stability of most of our IgGs is insensitive to pH, and at low ionic strength (≤0.15M), all IgG solutions are much more stable at pH 5 than at pH 7. In addition, the PEG-induced depletion force is less efficient in causing phase separation at pH 5 than at pH 7. In contrast to the native inter-protein interaction of IgGs, the effect of depletion force on phase separation of the antibody solutions is insensitive to ionic strength. Our results suggest that the long-range electrostatic inter-protein repulsion at low ionic strength stabilizes the IgG solutions at low pH. At high ionic strength, the short-range electrostatic interactions do not make a significant contribution to the colloidal stability for most IgGs with a few exceptions. The weaker effect of depletion force at lower pH indicates a reduction of protein concentration in the condensed phase. This work advances our basic understanding of the colloidal stability of IgG solutions and also introduces a practical approach to measuring protein colloidal stability under various solution conditions.

Concentration-dependent changes in apparent diffusion coefficients as indicator for colloidal stability of protein solutions

The colloidal stability of a protein solution during downstream processing, formulation, and storage is a key issue for the biopharmaceutical production process. Thus, knowledge about colloidal solution characteristics, such as the tendency to form aggregates or high viscosity, at various processing conditions is of interest. This work correlates changes in the apparent diffusion coefficient as a parameter of protein interactions with observed protein aggregation and dynamic viscosity of the respective protein samples. For this purpose, the diffusion coefficient, the protein phase behavior, and the dynamic viscosity in various systems containing the model proteins α-lactalbumin, lysozyme, and glucose oxidase were studied. Each of these experiments revealed a wide range of variations in protein interactions depending on protein type, protein concentration, pH, and the NaCl concentration. All these variations showed to be mirrored by changes in the apparent diffusion coefficient in the respective samples. Whereas stable samples with relatively low viscosity showed an almost linear dependence, the deviation from the concentration-dependent linearity indicated both an increase in the sample viscosity and probability of protein aggregation. This deviation of the apparent diffusion coefficient from concentration-dependent linearity was independent of protein type and solution properties for this study. Thus, this single parameter shows the potential to act as a prognostic tool for colloidal stability of protein solutions.

Improved heat stability of protein solutions and O/W emulsions upon dry heat treatment of whey protein isolate in the presence of low-methoxyl pectin

Whereas Whey Protein Isolate may be used as an effective emulsifier, it suffers from limited stability upon heating. In this research, the effect of combining Whey Protein Isolate (WPI) with Low Methoxyl Pectin (LMP) on the heat stability of WPI was investigated. This was accomplished either by simple mixing or by conjugate formation by dry heat treatment. WPI-LMP mixtures and conjugates were prepared at a WPI to LMP ratio of 1:0, 4:1, 2:1, and 1:1. Conjugates were prepared by means of dry heat treatment at a temperature of 60°C and 74% relative humidity by incubation for up to 16 days.The pH effect and brown color development of the WPI-LMP conjugates upon incubation was monitored. SDS-Page, free amino group determination, as well as diffusion coefficient analysis, all confirmed the formation of new compounds with high molecular weight. The heat stability of the conjugates was then tested and compared to the native WPI. Upon 2min of heating at 80°C and pH 6.5, the solubility of WPI was reduced by approximately 50% due to thermal denaturation and subsequent aggregation. However, dry heat treatment of the WPI-LMP mixtures highly improved the heat stability of WPI: as the incubation time was increased, the residual protein solubility upon heating of the WPI became higher. Upon 16 days of incubation, the protein solubility of the heated WPI-LMP conjugates was comparable to that of the conjugates before heating. Considering the emulsifying properties, it was found that WPI-LMP conjugates produced smaller oil droplet compared to either native WPI, dry heated WPI, or mixtures of WPI-LMP which were not subjected to conjugation by dry heat treatment. Heating the emulsions at 80°C for 10 and 20min revealed that WPI-LMP conjugates stabilized emulsions exhibited excellent stability towards heat: whereas pronounced aggregation and gelation occurred in emulsions stabilized by WPI or mixtures with LMP, the conjugate stabilized emulsions retained their original viscosity and particle size.

Protein folding and stability in the presence of osmolytes

Osmolytes are molecules with the function among others to align hydrostatic pressure between intracellular and extracellular spaces. Accumulation of osmolytes occurs in the cell in response to stress caused by pressure change, change in temperature, pH, and concentration of inorganic salts. Osmolytes can prevent native proteins denaturation and promote folding of unfolding proteins. Investigation of the osmolytes effect on these processes is essential for understanding the mechanisms of folding and functioning of proteins in vivo. A score of works, devoted to the effect of osmolytes on proteins, are not always consistent with each other. In this review an attempt was made to systemize available array of data on the subject and consider the problem of folding and stability of proteins in solutions in the presence of osmolytes from the single viewpoint.