Protein-salt Interactions Research Articles

Statistical thermodynamics of casein aggregation: Effects of salts and water

Salts, when added to milk, profoundly influence casein aggregation. Even though this well-known phenomenon has been widely exploited, there are still many unanswered questions. How do salts affect casein aggregation? Does water contribute significantly to the aggregation change? The key to answering these questions comes from statistical thermodynamics, i.e. the principles of physics that can link macroscopic data to the collective behaviour of molecules. We present two theoretical approaches. A rigorous approach which demands far more measurements than reported hitherto; and an approximate, pragmatic approach. It bases on stoichiometric models (isodesmic model for aggregation equilibria and von Smoluchowski model for kinetics) that can yield information on protein-water and protein-salt interactions from ‘real-life’ experimental measurements on model systems available in a variety of formats. Using experimental data from the literature, casein aggregation, in the absence of κ-casein, has been shown to be modulated by protein-salt interaction, while the contribution from water structure changes has been shown to be negligible.

Calorimetric studies of interactions between low molecular weight salts and bovine serum albumin in water at pH values below and above the isoionic point

Isothermal titration calorimetry was used to determine the temperature and salt concentration dependence of the enthalpy of mixing, ΔmixH, of bovine serum albumin (BSA) in aqueous buffer solutions with several low molecular weight salts. Three buffers were used: acetate (pH=4.0), MOPS (7.2), and borate (9.2). Since the isoionic point of BSA is at pI ≈ 4.7, the net charge of BSA in acetate buffer was positive (≈ +20), while in the other two buffer solutions it was negative (≈−15 in MOPS and ≈−25 in borate). The majority of the recorded heat effects were exothermic, while only at pH=9.2 a weak endothermic effect upon mixing BSA with LiCl, NaCl, and KCl was observed. For all buffer solutions the absolute values of ΔmixH of sodium salts followed the order: NaCl < NaBr < NaNO3< NaI < NaSCN, which is the reverse Hofmeister series for anions. The magnitude of the effects was the largest in acetate buffer and decreased with an increasing pH value of the solution. While the effect of varying the anion of the added salts was strongly pronounced at all pH values, the effect of the cation (LiCl, NaCl, KCl, RbCl and CsCl salts) was weak. The most interesting feature of the results obtained for pH >pI was the fact that ΔmixH were considerably more sensitive to the anion (co-ion to the net BSA charge) than to the cation species. This indicated that anions interacted quite strongly with the BSA even at pH values where the net charge of the protein was negative. We showed that ΔmixH at high addition of salts correlated well with the enthalpy of hydration of the corresponding salt anion. This finding suggested, consistently with some previous studies, that a part of the exothermic contribution to ΔmixH originated from the hydration changes upon the protein–salt interaction. Theoretical analysis, based on the primitive model of highly asymmetric electrolyte solutions solved within the mean spherical approximation, was used to estimate Coulomb effects upon mixing.

Effect of Protein-Lipid-Salt Interactions on Sodium Availability in the Mouth and Consequent Perception of Saltiness: As Affected by Hydration in Powders.

There is a broad need to reformulate lower sodium food products without affecting their original taste. The present study focuses on characterizing the role of protein-salt interactions on the salt release in low-moisture systems and saltiness perception during hydration. Sodium release from freeze-dried protein powders and emulsion powders formulated at different protein/lipid ratios (5:0 to 1:4) were characterized using a chromatography column modified with a porcine tongue. Emulsion systems with protein structured at the interface were found to have faster initial sodium release rates and faster hydration and were perceived to have a higher initial salt intensity with a lower salty aftertaste. In summary, exposure of the hydrophilic segments of the interface-structured proteins in emulsions was suggested to facilitate hydration and release of sodium during dissolution of low-moisture powder samples.

Protein Diffusiophoresis and Salt Osmotic Diffusion in Aqueous Solutions

Diffusion of a solute can be induced by the concentration gradient of another solute in solution. This transport mechanism is known as cross-diffusion. We have investigated cross-diffusion in a ternary protein-salt-water system. Specifically, we measured the two cross-diffusion coefficients for the lysozyme-NaCl-water system at 25 °C and pH 4.5 as a function of protein and salt concentrations by Rayleigh interferometry. One cross-diffusion coefficient characterizes salt osmotic diffusion induced by a protein concentration gradient, and is related to protein-salt thermodynamic interactions as described by the theories of Donnan membrane equilibrium and protein preferential hydration. The other cross-diffusion coefficient characterizes protein diffusiophoresis induced by a salt concentration gradient, and is described as the difference between a preferential-interaction coefficient and a transport parameter. We first relate our experimental results to the protein net charge and the thermodynamic excess of water near the protein surface. We then extract the Stefan-Maxwell diffusion coefficient describing protein-salt interactions in water. We find that the value of this coefficient is negative, contrary to the friction interpretation of Stefan-Maxwell equations. This result is explained by considering protein hydration. Finally, protein diffusiophoresis is quantitatively examined by considering electrophoretic and hydration effects on protein migration and utilized to accurately estimate lysozyme electrophoretic mobility. To our knowledge, this is the first time that protein diffusiophoresis has been experimentally characterized and a protein-salt Stefan-Maxwell diffusion coefficient reported. This work represents a significant contribution for understanding and modeling the effect of concentration gradients in protein-salt aqueous systems relevant to diffusion-based mass-transfer technologies and transport in living systems.

On the role of salt type and concentration on the stability behavior of a monoclonal antibody solution

Protein–salt interactions regulate protein solubility and stability and in particular several protein related processes, such as salting-out and aggregation. Using an IgG2 monoclonal antibody as a model multi-domain therapeutic protein, we have investigated the salt effect on the reversible formation of protein clusters with small aggregation number. The oligomer formation has been quantified by size exclusion chromatography (SEC). It is found that the salt effect is strongly ion specific and pH dependent. In particular, at pH 3.0 only anions affect the aggregation propensity, while at pH 4.0 both anions and cations influence the aggregation rate. The ranking of the anion effect follows the Hofmeister series with the only exception of sulfate, while that of the cation effect does not. In addition, a maximum of the aggregation propensity as a function of salt concentration is observed (i.e., presence of re-stabilization).By correlating the aggregation kinetics to the experimental investigation of protein structure and surface energy, it is shown that changes in pH and salt concentration induce aggregation not only through charge screening and various solvation forces, but also through the formation of protein intermediates characterized by partially ordered structures and certain degrees of hydrophobicity. The complex interaction between the solvation forces and such protein secondary structures induced by salts explains the observed experimental results relative to re-stabilization at large salt concentrations, ion specificity and the peculiar behavior of the sulfate anion.

Ions and the Protein Surface Revisited: Extensive Molecular Dynamics Simulations and Analysis of Protein Structures in Alkali-Chloride Solutions

Proteins interact with ions in various ways. The surface of proteins has an innate capability to bind ions, and it is also influenced by the screening of the electrostatic potential owing to the presence of salts in the bulk solution. Alkali metal ions and chlorides interact with the protein surface, but such interactions are relatively weak and often transient. In this paper, computer simulations and analysis of protein structures are used to characterize the interactions between ions and the protein surface. The results show that the ion-binding properties of protein residues are highly variable. For example, alkali metal ions are more often associated with aspartate residues than with glutamates, whereas chlorides are most likely to be located near arginines. When comparing NaCl and KCl solutions, it was found that certain surface residues attract the anion more strongly in NaCl. This study demonstrates that protein-salt interactions should be accounted for in the planning and execution of experiments and simulations involving proteins, particularly if subtle structural details are sought after.

The stabilities of protein crystals.

We describe a model for protein crystallization equilibria. The model includes four terms, (1) protein translational entropy opposes crystallization, (2) proteins are attracted to each other by a nonelectrostatic contact free energy favoring crystallization, (3) proteins in the crystal repel each other but, to a greater extent, attract counterions sequestered in the crystal, which favors crystallization, and (4) the translational entropy of the counterions opposes their sequestration into the crystal, opposing crystallization. We treat the electrostatics using the nonlinear Poisson-Boltzmann equation, and we use unit cell information from native protein crystals to determine the boundary conditions. This model predicts the stabilities of protein crystals as functions of temperature, pH, and salt concentrations, in good agreement with the data of Pusey et al. on tetragonal and orthorhombic crystal forms of lysozyme. The experiments show a weak dependence of crystal solubility on pH. According to the model, this is because the entropic cost to neutralize the crystal is compensated by favorable protein-salt interactions. Experiments also show that adding salt stabilizes the crystal. Cohn's empirical law predicts that the logarithm of solubility should be a linear function of salt. The present theory predicts nonlinearity, in better agreement with the experiments. The model shows that the salting out phenomena is not due to more counterion shielding but to lowered counterion translational entropy. Models of this type may help guide faster and better ways to crystallize proteins.

Charge‐dependent and charge‐independent contributions to ion‐protein interaction

The thermodynamic framework and recent molecular descriptions of protein-salt interactions are critically reviewed. A reanalysis of earlier and recent data describing the role of salts on the thermal stability of collagen I over a wide range of concentration evidences the complex encroachment of charge-dependent and charge-independent interactions. Charge-independent interactions, operationally quantified by dilution parameters and association constants, are found to be the overriding factor for the large stabilization observed with nonbinding salts and for the large destabilization observed when strong salt binding occurs. Charge-dependent interactions, namely screening and selective ion adsorption, are instead the prevailing components at low ionic strength and nonisoelectric pH.

Reproducible and efficient separation of aggregatable zein proteins by CZE using a volatile background electrolyte

Zein proteins are a complex mixture of polypetides that belong to the alcohol-soluble storage proteins group (prolamines) in corn. These proteins constitute about 50-60% of the total endosperm protein and are classified in different groups on the basis of differences in their solubility and sequence. Among them, zein proteins are considered the majority group showing a high tendency to aggregate what makes their analysis by any analytical method very difficult. Thus, CZE of these proteins requires the use of very complex BGEs noncompatible with online ESI-MS analysis. The aim of this work was to find a new BGE for the CZE separation of zein protein fully compatible with ESI-MS while providing further light on the complex CZE separation of aggregatable proteins. Thus, it is demonstrated in this work that efficient and reproducible CZE separations of zein proteins can be achieved by using a BGE composed of water, ACN, formic acid and ammonium hydroxide. Besides, it is shown that zein analysis is significantly improved by including the effect of an ammonium gradient during their separation. It is experimentally verified that the ammonium gradient can easily be achieved in CZE by either working with a sample zone with a low concentration of ammonium and a BGE with a high concentration, or conversely, working with a sample zone with high ammonium concentration and a BGE with low concentration of ammonium, giving rise in both cases to a significant improvement in the CZE separation of these proteins. It is demonstrated that this procedure can give rise to efficiency improvements of up to 20-fold in the CZE separation of zein proteins. Under optimized conditions, 20 proteins could be separated with average efficiencies higher than 400 000 theoretical plates/m. Some possible explanations of this effect are discussed including stacking, protein-capillary wall adsorption, protein solubility and protein-salt interactions.

A molecular-thermodynamic model for the interactions between globular proteins in aqueous solutions: Applications to bovine serum albumin (BSA), lysozyme, α-chymotrypsin, and immuno-gamma-globulins (IgG) solutions

To investigate globular protein–protein and protein–salt interactions in electrolyte solutions, a potential of mean force including hard-core repulsion, van der Waals attraction and electric double layer repulsion is proposed in this work. Both van der Waals attraction and double-layer repulsion are represented using hard spheres with two-Yukawa tails. The explicit analytical solution of osmotic pressure is derived from the first-order mean spherical approximation. From the comparison between the calculated and experimental values of osmotic pressures for aqueous bovine serum albumin (BSA), lysozyme, α-chymotrypsin, and immuno-gamma-globulins (IgG) solutions, we found that the proposed model is adequate for the description of the interactions between proteins at low ionic strength and small self-association of protein molecules. At high ionic strength, the charge inversions of protein molecules should be taken into account.

The Hofmeister series and protein-salt interactions

In order to understand the origin of the Hofmeister series, a statistical-mechanical analysis, based upon the Kirkwood-Buff (KB) theory, has been performed to extract information regarding protein hydration and water-mediated protein-salt interactions from published experimental data-preferential hydration and volumetric data for bovine serum albumin in the presence of a wide range of salts. The analysis showed a linear correlation between the preferential hydration parameter and the protein-cosolvent KB parameter. The same linear correlation holds even when nonelectrolyte cosolvents, such as polyethelene glycol, have been incorporated. These results suggest that the Hofmeister series is due to a wide variation of the water-mediated protein-cosolvent interaction (but not the change of protein hydration) and that this mechanism is a special case of a more general scenario common even to the macromolecular crowding.

A molecular approach to bioseparations: Protein–protein and protein–salt interactions

Many bioprocess separations involve manipulating the solution conditions to selectively remove or concentrate a target protein. The selectivity is determined by the thermodynamics of the protein solution, which are governed, at the molecular level, by protein–protein interactions. Thus, to optimise these processes, one must understand the precise nature of these interactions, how they are affected by the control parameters (e.g., temperature, ionic strength, pH), and how they relate to the solution properties of interest (e.g., protein solubility, protein stability, crystal nucleation rates). Recently, studies of protein–protein interactions have been motivated by the discovery of a crystallisation window, which places bounds on the protein–protein attraction that is required for crystallisation to be possible. This review focuses on experimental and theoretical studies of protein–protein interactions and discusses the current gaps in understanding these forces. The first models for these interactions had been based on DLVO theory, which has proven useful for understanding protein–protein interactions in dilute electrolyte solutions. However, DLVO theory does not include such important effects as anisotropic interactions, solvation forces, and specific ion effects. Various approaches have been developed to include these phenomena although they are still not well understood. One outstanding issue concerns specific ion effects, which play a crucial role in biological systems. An initial step in understanding these effects might be to first rationalize protein–salt interactions.

Electrolytes at spherical dielectric interfaces

A variational theory is developed and applied to study the properties of dielectric spheres immersed in a symmetric electrolyte solution. In the limit that the radius of the sphere becomes much larger than the Debye screening length, the system reduces to that of a planar dielectric interface. For this case, the excess surface tension obtained by the variational theory reduces to the Onsager-Samaras [J. Chem. Phys. 2, 528 (1934)] limiting law at low electrolyte concentrations. As the radius of the dielectric sphere decreases, the excess surface tension also decreases. The implications of this work to protein-salt interactions and the salting out of proteins are discussed.

Effects of pH, Salt, and Macromolecular Crowding on the Stability of FK506-binding Protein: An Integrated Experimental and Theoretical Study

Environmental variables can exert significant influences on the folding stability of a protein, and elucidating these influences provides insight on the determinants of protein stability. Here, experimental data on the stability of FKBP12 are reported for the effects of three environmental variables: pH, salt, and macromolecular crowding. In the pH range of 5-9, contribution to the pH dependence of the unfolding free energy from residual charge-charge interactions in the unfolded state was found to be negligible. The negligible contribution was attributed to the lack of sequentially nearest neighboring charged residues around groups that titrate in the pH range. KCl lowered the stability of FKBP12 and the E31Q/D32N double mutant at small salt concentrations but raised stability after approximately 0.5 M salt. Such a turnover behavior was accounted for by the balance of two opposing types of protein-salt interactions: the Debye-Hückel type, modeling the response of the ions to protein charges, favors the unfolded state while the Kirkwood type, accounting for the disadvantage of the ions moving toward the low-dielectric protein cavity from the bulk solvent, disfavors the unfolded state. Ficoll 70 as a crowding agent was found to have a modest effect on protein stability, in qualitative agreement with a simple model suggesting that the folded and unfolded states are nearly equally adversely affected by macromolecular crowding. For any environmental variable, it is the balance of its effects on the folded and unfolded states that determines the outcome on the folding stability.

Natural protein engineering: a uniquely salt-tolerant, but not halophilic, -type carbonic anhydrase from algae proliferating in low- to hyper-saline environments

Dunaliella salina is a unicellular green alga thriving in environments ranging from fresh water to hyper-saline lakes, such as the Dead Sea. An unusual, internally duplicated, 60 kDa alpha-type carbonic anhydrase (dCA I), located on the surface of this alga, is expected to function over a broad range of salinities. It would therefore differ from other carbonic anhydrases that already lose activity at low salinities and also from halophilic proteins that require high salinities for conformational stability. Enzymatic analyses indeed indicated that dCA I retained activity at salt concentrations ranging from low salt to at least 1.5 M NaCl or KCl for CO(2) hydration, 2.0 M NaCl for esterase activity and 0.5 M for bicarbonate dehydration. Although measurements at higher salinities were constrained by the interference of salt in the respective assayed reactions, activity was noticeable even at 4.0 M NaCl. Comparisons of the internally duplicated dCA I to single-domain derivatives indicated that inter-domain interactions played a decisive role in the stability, activity, salt tolerance and pH responses of dCA I. Hence dCA I is a uniquely salt- tolerant protein, retaining an active conformation over a large range of salinities and, as a Zn metalloenzyme, largely immune to the specific inhibitory effects of anions. Its unique features make dCA I a useful model to understand the physico-chemical basis of halotolerance and protein-salt interactions in general.

Concentration dependencies of NaCl salting of lysozyme by calorimetric methods

Concentration dependence of NaCl salting of lysozyme was investigated in the range of 0.5–9 mM lysozyme concentration in 0.1 M sodium acetate buffer, pH=4.25and the concentration of NaCl up to 0.1 M. Calorimetric experiments were performed with the use of a titration ITC Omega MicroCal calorimeter. It was found that the estimated number of bonding sites depended on the lysozyme concentration. For infinitely diluted lysozyme solution, the number of binding sites could be roughly estimated to ∼50. In the range of 2–9 mM protein concentration, the number of weakly binding ( K=2.7±0.8 M −1) sites on the protein surface was estimated to 35±7. McMillan and Mayer’s approach reduced to the third order virial coefficients demonstrates that besides the dominating effect of the protein—salt interaction ( a 11) the coefficient describing the lysozyme aggregation upon salt addition ( a 12) is statistically significant.

Link between protein-solvent and weak protein-protein interactions gives insight into halophilic adaptation.

Malate dehydrogenase (Hm MalDH) from the extreme halophile Haloarcula marismortui is a very acidic protein with extensive ion binding properties. It is a good model for the study of solvation-solubility relationships. We measured the small-angle neutron or X-ray scattering profiles of folded and stable Hm MalDH at various protein concentrations and derived the second virial coefficients A(2). In NaCl, CsCl, KF, KCl, and NaCH(3)CO(2), A(2) values are positive, indicating globally repulsive protein-protein interactions. Below 1 M MgCl(2) and MgSO(4) or above 2 M (NH(4))(2)SO(4), A(2) rapidly decreases. From structure factor modeling with DLVO (Derjaguin, Landau, Verwey, and Overbeek)-like potentials, an effective diameter of 80-82 A is found for the protein particle in solution, compatible with its structural dimensions; the effective charge of the particle is undefined because of the high salt concentration. The strong variations of the protein-protein interaction are correlated to an attractive potential whose depth evolves with the salinity but in an opposite way in Mg salts and (NH(4))(2)SO(4). A repulsive Donnan term, corresponding to counterion dissociation, and an attractive term related to previously measured preferential salt binding parameters are discussed from well-established thermodynamics considerations and qualitatively account for the behavior of the protein-protein interactions in the various solutions. Because a solvation shell with a composition different from bulk induces protein-protein attraction, molecular adaptation to high salt would be directed to allow protein-salt interactions in order to avoid water or salt enrichment at the surface of the protein and thus preserve its solubility.

Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes

Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutions of ammonium sulfate, sodium chloride, potassium isothiocyanate, and potassium chloride. The two-body interactions between ovalbumin molecules in concentrated ammonium-sulfate solutions can be described by the DLVO potentials plus a potential that accounts for the decrease in free volume available to the protein due to the presence of the salt ions. The interaction between ovalbumin and ammonium sulfate is unfavorable, reflecting the kosmotropic nature of sulfate anions. Lysozyme-lysozyme interactions cannot be described by the above potentials because anion binding to lysozyme alters these interactions. Lysozyme-isothiocyanate complexes are strongly attractive due to electrostatic interactions resulting from bridging by the isothiocyanate ion. Lysozyme-lysozyme interactions in sulfate solutions are more repulsive than expected, possibly resulting from a larger excluded volume of a lysozyme-sulfate bound complex or perhaps, hydration forces between the lysozyme-sulfate complexes.

Protein‐protein and protein‐salt interactions in aqueous protein solutions containing concentrated electrolytes

Biotechnology and BioengineeringVolume 58, Issue 4 p. 451-451 ErratumFree Access Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes R. A. Curtis, Corresponding Author R. A. Curtis Department of Chemical Engineering, University of California, Berkeley, Berkeley, California 94720Department of Chemical Engineering, University of California, Berkeley, Berkeley, California 94720Search for more papers by this authorA. Montaser, A. Montaser Department of Chemical Engineering, University of California, Berkeley, Berkeley, California 94720Search for more papers by this authorJ. M. Prausnitz, J. M. Prausnitz Chemical Sciences Division, Lawrence Berkeley Laboratory, University of California, Berkeley, Berkeley, California 94720Search for more papers by this authorH. W. Blanch, H. W. Blanch blanch@cchem.berkeley.edu Department of Chemical Engineering, University of California, Berkeley, Berkeley, California 94720Search for more papers by this author R. A. Curtis, Corresponding Author R. A. Curtis Department of Chemical Engineering, University of California, Berkeley, Berkeley, California 94720Department of Chemical Engineering, University of California, Berkeley, Berkeley, California 94720Search for more papers by this authorA. Montaser, A. Montaser Department of Chemical Engineering, University of California, Berkeley, Berkeley, California 94720Search for more papers by this authorJ. M. Prausnitz, J. M. Prausnitz Chemical Sciences Division, Lawrence Berkeley Laboratory, University of California, Berkeley, Berkeley, California 94720Search for more papers by this authorH. W. Blanch, H. W. Blanch blanch@cchem.berkeley.edu Department of Chemical Engineering, University of California, Berkeley, Berkeley, California 94720Search for more papers by this author First published: 26 March 2000 https://doi.org/10.1002/(SICI)1097-0290(19980520)58:4<451::AID-BIT13>3.0.CO;2-ECitations: 7AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume58, Issue420 May 1998Pages 451-451 RelatedInformation

Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes.

Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutions of ammonium sulfate, sodium chloride, potassium isothiocyanate, and potassium chloride. The two-body interactions between ovalbumin molecules in concentrated ammonium-sulfate solutions can be described by the DLVO potentials plus a potential that accounts for the decrease in free volume available to the protein due to the presence of the salt ions. The interaction between ovalbumin and ammonium sulfate is unfavorable, reflecting the kosmotropic nature of sulfate anions. Lysozyme-lysozyme interactions cannot be described by the above potentials because anion binding to lysozyme alters these interactions. Lysozyme-isothiocyanate complexes are strongly attractive due to electrostatic interactions resulting from bridging by the isothiocyanate ion. Lysozyme-lysozyme interactions in sulfate solutions are more repulsive than expected, possibly resulting from a larger excluded volume of a lysozyme-sulfate bound complex or perhaps, hydration forces between the lysozyme-sulfate complexes.