Patterns of protein protein interactions in salt solutions and implications for protein crystallization

The second osmotic virial coefficients of seven proteins-ovalbumin, ribonuclease A, bovine serum albumin, alpha-lactalbumin, myoglobin, cytochrome c, and catalase-were measured in salt solutions. Comparison of the interaction trends in terms of the dimensionless second virial coefficient b(2) shows that, at low salt concentrations, protein-protein interactions can be either attractive or repulsive, possibly due to the anisotropy of the protein charge distribution. At high salt concentrations, the behavior depends on the salt: In sodium chloride, protein interactions generally show little salt dependence up to very high salt concentrations, whereas in ammonium sulfate, proteins show a sharp drop in b(2) with increasing salt concentration beyond a particular threshold. The experimental phase behavior of the proteins corroborates these observations in that precipitation always follows the drop in b(2). When the proteins crystallize, they do so at slightly lower salt concentrations than seen for precipitation. The b(2) measurements were extended to other salts for ovalbumin and catalase. The trends follow the Hofmeister series, and the effect of the salt can be interpreted as a water-mediated effect between the protein and salt molecules. The b(2) trends quantify protein-protein interactions and provide some understanding of the corresponding phase behavior. The results explain both why ammonium sulfate is among the best crystallization agents, as well as some of the difficulties that can be encountered in protein crystallization.     

A comparative study of monoclonal antibodies. 1. phase behavior and protein–protein interactions

Protein phase behavior is involved in numerous aspects of downstream processing, either by design as in crystallization or precipitation processes, or as an undesired effect, such as aggregation. This work explores the phase behavior of eight monoclonal antibodies (mAbs) that exhibit liquid–liquid separation, aggregation, gelation, and crystallization. The phase behavior has been studied systematically as a function of a number of factors, including solution composition and pH, in order to explore the degree of variability among different antibodies. Comparisons of the locations of phase boundaries show consistent trends as a function of solution composition; however, changing the solution pH has different effects on each of the antibodies studied. Furthermore, the types of dense phases formed varied among the antibodies. Protein–protein interactions, as reflected by values of the osmotic second virial coefficient, are used to correlate the phase behavior. The primary findings are that values of the osmotic second virial coefficient are useful for correlating phase boundary locations, though there is appreciable variability among the antibodies in the apparent strengths of the intrinsic protein–protein attraction manifested. However, the osmotic second virial coefficient does not provide a clear basis to predict the type of dense phase likely to result under a given set of solution conditions. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:268–276, 2015     

From osmotic second virial coefficient (B22) to phase behavior of a monoclonal antibody

Antibodies are complex macromolecules and their phase behavior as well as interactions within different solvents and precipitants are still not understood. To shed some light into the processes on a molecular dimension, the occurring self‐interactions between antibody molecules were analyzed by means of the osmotic second virial coefficient (B₂₂). The determined B₂₂ follows qualitatively the phenomenological Hofmeister series describing the aggregation probability of antibodies for the various solvent compositions. However, a direct correlation between crystallization probability and B₂₂ in form of a crystallization slot does not seem to be feasible for antibodies since the phase behavior is strongly dependent on their anisotropy. Kinetic parameters have to be taken into account due to the molecular size and complexity of the molecules. This is confirmed by a comparison of experimental data with a theoretical phase diagram. On the other hand the solubility is thermodynamically driven and therefore the B₂₂ could be used to establish a universal solubility line for the monoclonal antibody mAb04c and different solvent compositions by using thermodynamic models. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:438–451, 2015     

Interactions and phase behavior of a monoclonal antibody

Protein phase behavior is implicated in numerous aspects of downstream processing either by design, as in crystallization or precipitation processes, or as an undesired effect, such as aggregation. An improved understanding of protein phase behavior is, therefore, important for developing rational design strategies for important process steps. This work explores the phase behavior of a monoclonal antibody (mAb), IDEC-152, which exhibits liquid-liquid separation, aggregation, gelation, and crystallization. A systematic study of numerous factors, including the effects of solution composition and pH, has been conducted to explore the phase behavior of this antibody. Phenomena observed include a significant dependence of the cloud point on the cation in sulfate salts and nonmonotonic trends in pH dependence. Additionally, conditions for crystallization of this mAb are reported for the first time. Protein-protein interactions, as determined from the osmotic second virial coefficient, are used to interpret the phase behavior.     

Specific Ions Effect on Emulsions, Foams, and Gels of a Seed Protein

A protein concentrate was prepared from the seeds of jack bean (Canavalia ensiformis), and the influence of selected Hofmeister salts on functional properties of the protein concentrate was investigated. Results indicated that kosmotropic salts (Na₂SO₄, NaCl, NaBr) improved water absorption capacity and least gelation concentration of the proteins more than chaotropic salts (NaI, NaClO₄, NaSCN) did. The reduction in water absorption capacity followed the Hofmeister trend (Na₂SO₄ > NaCl > NaBr > NaI > NaClO₄ > NaSCN). However, a reverse trend was observed with the foaming and emulsifying properties. These findings are insightful in understanding the structure–property relations of the concentrate.     

Relative effectiveness of various anions on the solubility of acidic Hypoderma lineatum collagenase at pH 7.2.

The effects of various anions on decreasing the solubility of acidic Hypoderma lineatum collagenase at pH 7.2 and 18 degrees C were qualitatively defined by replacing the crystallizing agent of known crystallization conditions by various ammonium salts. The solubility curves measured in the presence of the sulfate, phosphate, citrate, and chloride ammonium salts gave the following ranking of anions: HPO4(2-)/H2PO4- > SO4(2-) > citrate 3-/citrate2- >> Cl-. This order is in agreement with the Hofmeister series. In a previous study on the solubility at pH 4.5 of lysozyme, a basic protein, the effectiveness of anions in decreasing the solubility was found to be in the reverse order. This suggests that the effectiveness of anions in the crystallization of proteins is dependent on the net charge of the protein, i.e., depending on whether a basic protein is crystallized at acidic pH or an acidic protein at basic pH.     

Influence of precipitating agents on thermodynamic parameters of protein crystallization solutions

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.     

Protein unfolding during reversed-phase chromatography: II. Role of salt type and ionic strength.

While reversed-phase chromatography (RPC) may be a powerful method for purification of proteins at the analytical scale, both preparative and analytical applications have been hindered by the complex chromatographic behavior of proteins compared to small molecules. Further, preparative applications have been limited because of poor yields caused by the denaturing conditions involved. One means for modulating both the stability and chromatographic behavior of proteins is through the use of added salt. In this investigation, we show how salt type and ionic strength affect protein conformation on RPC surfaces. Exposure of amide groups of adsorbed BPTI was monitored using nuclear magnetic resonance (NMR) spectroscopy and hydrogen-deuterium isotope exchange. Sodium chloride, sodium acetate, and ammonium sulfate were studied at ionic strengths up to I = 0.375, with adsorption hold times being 5 min and 2 h. We found that increasing ionic strength decreased exposure of the exchange reporter groups in essentially all cases. However, even at the same ionic strength the level and distribution of residue protection varied with salt type and hold time. NaCl does not protect certain reporter groups at all, while those that it does protect to some degree at short hold times can exchange slightly more at longer times. The pattern and level of protection for NaAc at short times is similar to that for NaCl, but at longer times more uniform protection is seen as the reporter groups completely exposed at short times become more protected. For (NH(4))(2)SO(4) the pattern of protection at short hold time is similar to those of the other salts, although it protects all groups much more. This would be expected from the Hofmeister series. However, at longer times the level of protection with (NH(4))(2)SO(4) decreases below that of the other salts, while it uniquely protects all groups to nearly the same level. Such subtle variations in the protein structure would not have been detected without the measurements and analysis used here. Chromatographic retention times and peak shapes were obtained for the above systems. Variations of behavior were seen that could not be correlated with any of the above protection patterns and levels or even with heuristics such as the Hofmeister series. This suggests further conformational changes upon elution may be critical to the retention process. However, an excellent correlation was found between peak width at half-height and the average degree of unfolding, as indicated by the average level of isotopic exchange. Thus, while further studies are needed to definitively determine the connection between protein unfolding and retention, use of this correlation may improve designing and screening for chromatographic conditions that minimize protein unfolding.     

Effect of glycosylation on the partition behavior of a human antibody in aqueous two‐phase systems

Human proteins are expressed in some hosts wrongly glycosylated or nonglycosylated. Although it is accepted that glycosylation contributes to the stability of the protein in solution, the effect of glycosylation on the stability of human antibodies is not fully understood. In this work, we present solubility studies of two human antibodies that have the same primary structure but different glycosylation pattern. The studies were done by monitoring the partitioning behavior of both proteins in a series of aqueous two‐phase systems at and away the isoelectric point of the proteins and at different temperatures. Our studies show that in the absence of direct electrostatic forces, the partitioning behavior of the antibodies depends on the presence or absence of the polysaccharide chains. Overall, the nonglycosylated protein is less soluble than the glycosylated one. The potential of aqueous two‐phase systems for the separation of the glycosylated and nonglycosylated proteins was also explored. A simple series of extractions seems to be enough to separate the glycosylated variety from the nonglycosylated one at high purity but low yields. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:943–950, 2013     

Anion Effect on the Binary and Ternary Phase Diagrams of Chiral Medetomidine Salts and Conglomerate Crystal Formation

The binary phase diagrams of hydrogen halides salts of medetomidine (Med.HX, X:Br,I) and hydrogen oxalate salt of medetomidine (Med.Ox) were determined based on thermogravimetric/differential thermal analysis (TGA/DTA) and their crystal structure behavior was confirmed by comparison of the X‐ray diffractometry and FT‐IR spectroscopy of the racemate and pure enantiomer. All hydrogen halide salts presented racemic compound behavior. Heat of fusion of halides salt of (rac)‐medetomidine decreased with ionic radius increase. Eutectic points for Med.HCl (previously reported), Med.HBr, and Med.HI rest were unchanged approximately. The solubility of different enantiomeric mixtures of Med.HBr and Med.HI were measured at 10, 20, and 30°C in 2‐propanol showing a solubility increase with ionic radius. A binary phase diagram of Med.Ox shows a racemic conglomerate behavior. The solubility of enantiomeric mixtures of Med.Ox were measured at 10, 20, 30, and 40°C. The ternary phase diagram of Med.Ox in ethanol conforms to a conglomerate crystal forming system, favoring its enantiomeric purification by preferential crystallization. Chirality 26:183–188, 2014. © 2014 Wiley Periodicals, Inc.     

Some characteristics of protein precipitation by salts

The solubilities of lysozyme, alpha-chymotrypsin and bovine serum albumin (BSA) were studied in aqueous electrolyte solution as a function of ionic strength, pH, the chemical nature of salt, and initial protein concentration. Compositions were measured for both the supernatant phase and the precipitate phase at 25 degrees C. Salts studied were sodium chloride, sodium sulfate, and sodium phosphate. For lysozyme, protein concentrations in supernatant and precipitate phases are independent of the initial protein concentration; solubility can be represented by the Cohn salting-out equation. Lysozyme has a minimum solubility around pH 10, close to its isoelectric point (pH 10.5). The effectiveness of the three salts studied for precipitation were in the sequence sulfate > phosphate > chloride, consistent with the Hofmeister series. However, for alpha-chymotrypsin and BSA, initial protein concentration affects the apparent equillibrium solubility. For these proteins, experimental results show that the compositions of the precipitate phase are also affected by the initial protein concentration. We define a distribution coefficient kappa(e) to represent the equilibrium ratio of the protein concentration in the supernatant phase to that in the precipitate phase. When the salt concentration is constant, the results show that, for lysozyme, the protein concentrations in both phases are independent of the initial protein concentrations, and thus kappa(e) is a constant. For alpha-chymotrypsin and BSA, their concentrations in both phases are nearly proportional to the initial protein concentrations, and therefore, for each protein, at constant salt concentration, the distribution coefficient kappa(e) is independent of the initial protein concentration. However, for both lysozyme and alpha-chymotrypsin, the distribution coefficient falls with increasing salt concentration. These results indicate that care must be used in the definition of solubility. Solubility is appropriate when the precipitate phase is pure, but when it is not, the distribution coefficient better describes the phase behavior. (c) 1992 John Wiley & Sons, Inc.     

Ion-induced alterations of the local hydration environment elucidate Hofmeister effect in a simple classical model of Trp-cage miniprotein

Protein stability is known to be influenced by the presence of Hofmeister active ions in the solution. In addition to direct ion-protein interactions, this influence manifests through the local alterations of the interfacial water structure induced by the anions and cations present in this region. In our earlier works it was pointed out that the effects of Hofmeister active salts on the stability of Trp-cage miniprotein can be modeled qualitatively using non-polarizable force fields. These simulations reproduced the structure-stabilization and structure-destabilization effects of selected kosmotropic and chaotropic salts, respectively. In the present study we use the same model system to elucidate atomic processes behind the chaotropic destabilization and kosmotropic stabilization of the miniprotein. We focus on changes of the local hydration environment of the miniprotein upon addition of NaClO₄ and NaF salts to the solution. The process is separated into two parts. In the first, ‘promotion’ phase, the protein structure is fixed, and the local hydration properties induced by the simultaneous presence of protein and ions are investigated, with a special focus on the interaction of Hofmeister active anions with the charged and polar sites. In the second, ‘rearrangement’ phase we follow changes of the hydration of ions and the protein, accompanying the conformational relaxation of the protein. We identify significant factors of an enthalpic and entropic nature behind the ion-induced free energy changes of the protein-water system, and also propose a possible atomic mechanism consistent with the Collins’s rule, for the chaotropic destabilization and kosmotropic stabilization of protein conformation.     

The influence of kosmotropic and chaotropic salts on the functional properties of Mucuna pruriens protein isolate

The influence of chaotropic and kosmotropic salts on Mucuna pruriens protein isolates was investigated. Protein solubility profile indicated that solubility was minimal at the isoelectric point of the protein isolate (4.0) while the solubility was maximal at pH 10.0 in all salt solutions. Chaotropes (I(-), ClO(4)(-) and SCN(-)) exhibit better protein solubility than the kosmotropes (SO(4)(2-), Cl(-) and Br(-)). Increase in protein solubility follows the Hofmeister series: NaSO(4)相似文献   

The Hofmeister effect on amyloid formation using yeast prion protein

A variety of proteins are capable of converting from their soluble forms into highly ordered fibrous cross‐β aggregates (amyloids). This conversion is associated with certain pathological conditions in mammals, such as Alzheimer disease, and provides a basis for the infectious or hereditary protein isoforms (prions), causing neurodegenerative disorders in mammals and controlling heritable phenotypes in yeast. The N‐proximal region of the yeast prion protein Sup35 (Sup35NM) is frequently used as a model system for amyloid conversion studies in vitro. Traditionally, amyloids are recognized by their ability to bind Congo Red dye specific to β‐sheet rich structures. However, methods for quantifying amyloid fibril formation thus far were based on measurements linking Congo Red absorbance to concentration of insulin fibrils and may not be directly applicable to other amyloid‐forming proteins. Here, we present a corrected formula for measuring amyloid formation of Sup35NM by Congo Red assay. By utilizing this corrected procedure, we explore the effect of different sodium salts on the lag time and maximum rate of amyloid formation by Sup35NM. We find that increased kosmotropicity promotes amyloid polymerization in accordance with the Hofmeister series. In contrast, chaotropes inhibit polymerization, with the strength of inhibition correlating with the B‐viscosity coefficient of the Jones‐Dole equation, an increasingly accepted measure for the quantification of the Hofmeister series.     

Lysozyme-lysozyme self-interactions as assessed by the osmotic second virial coefficient: Impact for physical protein stabilization

The purpose of the presented study is to understand the physicochemical properties of proteins in aqueous solutions in order to identify solution conditions with reduced attractive protein-protein interactions, to avoid the formation of protein aggregates and to increase protein solubility. This is assessed by measuring the osmotic second virial coefficient (B²²), a parameter of solution non-ideality, which is obtained using self-interaction chromatography. The model protein is lysozyme. The influence of various solution conditions on B²² was investigated: protonation degree, ionic strength, pharmaceutical relevant excipients and combinations thereof. Under acidic solution conditions B²² is positive, favoring protein repulsion. A similar trend is observed for the variation of the NaCl concentration, showing that with increasing the ionic strength protein attraction is more likely. B²² decreases and becomes negative. Thus, solution conditions are obtained favoring attractive protein-protein interactions. The B²² parameter also reflects, in general, the influence of the salts of the Hofmeister series with regard to their salting-in/salting-out effect. It is also shown that B²² correlates with protein solubility as well as physical protein stability.     

Salts enhance both protein stability and amyloid formation of an immunoglobulin light chain

Amyloid fibrils are associated with sulfated glycosaminoglycans in the extracellular matrix. The presence of sulfated glycosaminoglycans is known to promote amyloid formation in vitro and in vivo, with the sulfate groups playing a role in this process. In order to understand the role that sulfate plays in amyloid formation, we have studied the effect of salts from the Hofmeister series on the protein structure, stability and amyloid formation of an amyloidogenic light chain protein, AL-12. We have been able to show for the first time a direct correlation between protein stability and amyloid formation enhancement by salts from the Hofmeister series, where SO₄²⁻ conferred the most protein stability and enhancement of amyloid formation. Our study emphasizes the importance of the effect of ions in the protein bound water properties and downplays the role of specific interactions between the protein and ions.     

The systematic characterization by aqueous column chromatography of solutes which affect protein stability

We have systematically characterized, by aqueous column chromatography on a size exclusion cross-linked dextran gel (Sephadex G-10), 12 solutes, 11 of which are known to affect protein stability. Six are chaotropes (water structure breakers) and destabilize proteins, while five are polar kosmotropes (polar water structure makers) and stabilize proteins. Analysis of the chromatographic behavior of these neutral (ethylene glycol, urea), positively charged (Tris, guanidine, as the hydrochloride salts) and negatively charged (SO2-4, HPO2-4, F-, Cl-, Br-, Cl3CCO-2, I-, SCN-, as the sodium salts, in order of elution) solutes at pH 7 as a function of sample concentration (up to 0.6 M), supporting electrolyte, and temperature yields four conclusions, based largely on the behavior of the anions. Chaotropes adsorb to the gel according to their position in the Hofmeister series, with the most chaotropic species adsorbing most strongly. ++Chaotropes adsorb to the gel less strongly in the presence of chaotropes (a salting in effect) and more strongly in the presence of polar kosmotropes (a salting out effect). Polar kosmotropes do not adsorb to the gel, and are sieved through the gel according to their position in the Hofmeister series, with the most kosmotropic species having the largest relative hydrodynamic radii. The hydrodynamic radii of polar kosmotropes is increased by chaotropes and decreased by polar kosmotropes. These results suggest that a chaotrope interacts with the first layer of immediately adjacent water molecules somewhat less strongly than would bulk water in its place; a polar kosmotrope, more strongly.     

Factors influencing protein structure during acid precipitation: A study of soya proteins

Summary The influence of mixing conditions and acid type upon soya protein structure during isoelectric precipitation have been investigated. The extent of protein modification after precipitation was dependent on the acid anion following the inverse of the Hofmeister series which classifies anions in decreasing order of effectiveness as precipitants. With the anion, SO ₄ ^2– , which caused least protein modification, mixing could be varied over the range of mixing Reynolds number 2,800 to 28,000 with only a small effect on protein structure. On the other hand, hydrochloric acid caused substantial damage at the lower end of this mixing range.     

The effects of mono- and divalent salts on the O2-evolution activity and low temperature multiline EPR spectrum of Photosystem II preparations from spinach

The ability of salts to inhibit the O2-evolution activity of PS II preparations is shown to parallel closely the Hofmeister series, suggesting that inhibition is related to the solubility of the 16, 24 and 33 kDa proteins in these salt solutions. An examination of the effect of salt inactivation on the low temperature multiline EPR signal indicates that the release of either the 16 and 24 kDa proteins, or additionally the 33 kDa protein blocks or greatly reduces the efficiency of the advancement of the water-splitting complex to the S2-state; under some conditions, this inhibition is reversible.