Activity coefficients of salts in highly concentrated protein solutions: I. Alkali chlorides in isoionic bovine serum albumin solutions

In order to understand the thermodynamic state of simple salts in living cells, the mean activity coefficients of LiCl, NaCl, KC1, RbCl, CsCl were determined in concentrated isoionic bovine serum albumin (BSA) solutions by use of the EMF method with ion exchange membrane electrodes. The protein concentration range extended up to 22 wt %, whereas the salt concentration was kept constant at 0.1 mole per kilogram water. These solutions may be regarded as crude but appropriate model systems for the cytoplasm of cells as far as type and magnitude of the macromolecular component influence on the chemical potential of the salts is concerned. The mean stoichiometric activity coefficients of the alkali chlorides in the isoionic BSA solutions decreased linearly with the protein molality; this decrease, however, did not exceed ca. 10% compared with the pure 0.1 molal salt solutions. Only very small differences in the behaviour of the different alkali chlorides were observed. The results may be interpreted by the superposition of the effects of specific Cl^? ion binding to BSA and BSA bound “non-solvent” water with probably electrostatic long range interactions of the BSA(Cl^?)_v polyions with the salt ions in solution. The resulting mean activity coefficients, corrected for ion binding and non-solvent water, showed a very slight linear dependence on the protein concentration. The departure from the value in the pure 0.1 molal salt solutions did not exceed ± 2%.     

Activity coefficients of KCl in highly concentrated protein solutions

As a contribution to the understanding of the thermodynamic state of single salts in living systems, the activity coefficients of KCl were determined in concentrated bovine serum albumin (BSA) solutions. The concentration range studied was 0.01 to 0.5 M KCl and zero to 18% wt BSA, thus amply covering physiological conditions. The activity coefficients of the salt were measured using the EMF method with ion exchange membrane electrodes. Keeping the salt concentration constant, the activity coefficients of the salt decrease linearly with protein concentration, the effect being more pronounced for low salt content. The maximal deviations of the activity coefficients with respect to those in pure salt solution amount to ca. 40% for 0.01 M KCl and 18% wt BSA. The results were interpreted on the assumption of the superposition of three effects i.e. water bound to BSA molecules as non-solvent water, specific Cl^– ion binding and the electrostatic interactions of the polyions with the salt ions. In view of the results it can be concluded that only a small portion of simple intracellular ions are bound, based on the assumption that the cytoplasm of living cells may be regarded as a concentrated protein-salt solution.     

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.     

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.     

Thermodynamics of interactions of urea and guanidinium salts with protein surface: relationship between solute effects on protein processes and changes in water-accessible surface area.

To interpret effects of urea and guanidinium (GuH(+)) salts on processes that involve large changes in protein water-accessible surface area (ASA), and to predict these effects from structural information, a thermodynamic characterization of the interactions of these solutes with different types of protein surface is required. In the present work we quantify the interactions of urea, GuHCl, GuHSCN, and, for comparison, KCl with native bovine serum albumin (BSA) surface, using vapor pressure osmometry (VPO) to obtain preferential interaction coefficients (Gamma(mu3)) as functions of nondenaturing concentrations of these solutes (0-1 molal). From analysis of Gamma(mu3) using the local-bulk domain model, we obtain concentration-independent partition coefficients K(nat)(P) that characterize the accumulation of these solutes near native protein (BSA) surface: K(nat)(P,urea)= 1.10 +/- 0.04, K(nat)(P,SCN(-)) = 2.4 +/- 0.2, K(nat)(P,GuH(+)) = 1.60 +/- 0.08, relative to K(nat)(P,K(+)) identical with 1 and K(nat)(P,Cl(-)) = 1.0 +/- 0.08. The relative magnitudes of K(nat)(P) are consistent with the relative effectiveness of these solutes as perturbants of protein processes. From a comparison of partition coefficients for these solutes and native surface (K(nat)(P)) with those determined by us previously for unfolded protein and alanine-based peptide surface K(unf)(P), we dissect K(P) into contributions from polar peptide backbone and other types of protein surface. For globular protein-urea interactions, we find K(nat)(P,urea) = K(unf)(P,urea). We propose that this equality arises because polar peptide backbone is the same fraction (0.13) of total ASA for both classes of surface. The analysis presented here quantifies and provides a physical basis for understanding Hofmeister effects of salt ions and the effects of uncharged solutes on protein processes in terms of K(P) and the change in protein ASA.     

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   

A circular dichroism study of charged polypeptides interaction with salts.

The effect of salts on the experimental circular dichroism spectra of polypeptides is presented using poly-L-lysine as the main model. Salt effects are analyzed into: (a) shielding at low (less than 0.5 M) concentrations of all salts; (b) binding to positively charged and some neutrally charged side-chains by certain anions (e.g., CCl3COO-, CF3C00-, ClO4-), with induction of helicity; (c) binding of these same anions, at high concentration, to the backbone leading toward random structure; (d) binding of high concentration of denaturing cations (La+3, Ca++, Li+) to the backbone, with La+3 and Ca++ leading to collapsed random structure (R) while Li+ tends to leave the polypeptide somewhat extended; (e) indirect interaction of salting-out salts (NaH2PO4, (NH4)2SO4, NH4F), at high concentration, leading toward complete alpha helicity, probably by competition with the polypeptide and the anion for available water. Effects of changing the temperature from 5 degrees to 50 degrees on the circular dishroism spectra of different polypeptide-salt solutions throughout the region from extended (LES) to alpha helical conformation are analyzed in terms of introduction of randomness (R) at high temperature. Applications to effects of salt on protein structures are considered.     

Compressibility,densimetric and calorimetric studies of hydration of carrageenans in the random form

The partial molal volume and adiabatic compressibility were measured, as well as their counterion activity, for sodium and potassium salts of three types of carrageenan (κ-, ι- and λ-components) in aqueous solutions at 25°C. Furthermore, the amount of related unfreezable water was estimated by the differential scanning calorimetry. On the basis of these results, the hydration states of carrageenans in the random form were comparatively discussed in relation to their chemical structure, counterion binding and polymer concentration. The sodium salt of each component showed a larger amount of hydration when compared with the corresponding potassium salt. The amount of hydration estimated from molal volume and compressibility data (in dilute solution) increased in the order of κ < ι < λ, while the amount of unfreezable water (in concentrated solution) decreased in the same order. These characteristics hydration behaviours of carrageenans seemed to be reasonably explained in terms of the effects of the charge density and counterion dissociation of these polyions.     

Radiation-induced chain reactions in alcohol solutions of diphenyliodonium salts: a high-sensitivity chemical dosimeter

Chain reactions in gamma-irradiated 2-propanol solutions of diphenyliodonium salts have been studied. Protonic acids were generated in the irradiated solutions with high yields, whereas acid formation as a result of thermal reactions was negligible. The solution can be used as a high-sensitivity chemical dosimeter. The G value of acidic protons increases with increasing concentration of diphenyliodonium salt at the lower concentrations because the reaction rate of a propagation reaction increases. However, the chain is limited by a termination reaction between phenyl radical and the iodonium salt: The G value shows a maximum value of 610 micromol J(-1) at the concentration of 0.08 mol dm(-3) and decreases at higher salt concentrations.     

Cloud-point temperatures for lysozyme in electrolyte solutions: effect of salt type, salt concentration and pH

Liquid-liquid phase-separation data were obtained for aqueous saline solutions of hen egg-white lysozyme at a fixed protein concentration (87 g/l). The cloud-point temperature (CPT) was measured as a function of salt type and salt concentration to 3 M, at pH 4.0 and 7.0. Salts used included those from mono and divalent cations and anions. For the monovalent cations studied, as salt concentration increases, the CPT increases. For divalent cations, as salt concentration rises, a maximum in the CPT is observed and attributed to ion binding to the protein surface and subsequent water structuring. Trends for sulfate salts were dramatically different from those for other salts because sulfate ion is strongly hydrated and excluded from the lysozyme surface. For anions at fixed salt concentration, the CPT decreases with rising anion kosmotropic character. Comparison of CPTs for pH 4.0 and 7.0 revealed two trends. At low ionic strength for a given salt, differences in CPT can be explained in terms of repulsive electrostatic interactions between protein molecules, while at higher ionic strength, differences can be attributed to hydration forces. A model is proposed for the correlation and prediction of the CPT as a function of salt type and salt concentration. NaCl was chosen as a reference salt, and CPT deviations from that of NaCl were attributed to hydration forces. The Random Phase Approximation, in conjunction with a square-well potential, was used to calculate the strength of protein-protein interactions as a function of solution conditions for all salts studied.     

Mean activity coefficients for the simple electrolyte in aqueous mixtures of polyelectrolyte and simple electrolyte. V. common counterion mixtures of alkali-metal dextransulfates with alkali metal chlorides

Mean molal activity coefficients of simple electrolyte in aqueous solutions of Li, Na, K or Cs salts of dextransulfate (DS) with added LjCl, NaCl, KCl or CsCl are reported. The measurements were carried out by means of an electrochemical cell method using a cation exchange membrane as cation selective electrode and Ag/AgCl electrodes. For LiDS-LiCl, NaDS-NaCl and CsDS-CsCl systems the polymer concentration, m_p, was varied from 0.0088 to 0.113 m and at a given m_p the ratio X of the polymer to salt concentration was varied from 0.5 to 16. Due to the insolubility of KDS in high concentration of KCl, the measurements on KDS-KCl system were performed in the m_p range of 0.0088–0.089 m and some of the smaller X values were omitted. The activity coefficient results are compared to Manning's limiting laws, the additivity rule, and to new limiting laws. The additivity rule can give an excellent representation of the data for all m_p values when γ_p is used as an adjustable parameter.     

Theoretical and experimental studies on freezing point depression and vapor pressure deficit as methods to measure osmotic pressure of aqueous polyethylene glycol and bovine serum albumin solutions

For survival in adverse environments where there is drought, high salt concentration or low temperature, some plants seem to be able to synthesize biochemical compounds, including proteins, in response to changes in water activity or osmotic pressure. Measurement of the water activity or osmotic pressure of simple aqueous solutions has been based on freezing point depression or vapor pressure deficit. Measurement of the osmotic pressure of plants under water stress has been mainly based on vapor pressure deficit. However, differences have been noted for osmotic pressure values of aqueous polyethylene glycol (PEG) solutions measured by freezing point depression and vapor pressure deficit. For this paper, the physicochemical basis of freezing point depression and vapor pressure deficit were first examined theoretically and then, the osmotic pressure of aqueous ethylene glycol and of PEG solutions were measured by both freezing point depression and vapor pressure deficit in comparison with other aqueous solutions such as NaCl, KCl, CaCl(2), glucose, sucrose, raffinose, and bovine serum albumin (BSA) solutions. The results showed that: (1) freezing point depression and vapor pressure deficit share theoretically the same physicochemical basis; (2) theoretically, they are proportional to the molal concentration of the aqueous solutions to be measured; (3) in practice, the osmotic pressure levels of aqueous NaCl, KCl, CaCl(2), glucose, sucrose, and raffinose solutions increase in proportion to their molal concentrations and there is little inconsistency between those measured by freezing point depression and vapor pressure deficit; (4) the osmotic pressure levels of aqueous ethylene glycol and PEG solutions measured by freezing point depression differed from the values measured by vapor pressure deficit; (5) the osmotic pressure of aqueous BSA solution measured by freezing point depression differed slightly from that measured by vapor pressure deficit.     

The effect of low ionic strength extracellular solutions on the resting potential in skeletal muscle fibers

Intracellular measurements of the resting potential were made in fibers of the frog sartorius muscle in solutions of varying salt composition and concentration to determine the effects of low ionic strength extracellular solutions on the resting potential. Changes in the glass microelectrode tip potential in low ionic strength solutions were minimized by adding ThCl₄ to the extracellular solution. These experimental conditions allowed measurement of the relationship of the resting potential to the concentration of the salt in the extracellular solution by replacing it with the nonionic substance, sucrose. Substitution of sucrose for the extracellular NaCl produced a stable depolarization which was logarithmically related to the NaCl concentration. Substitution of sucrose for choline Cl, instead of NaCl, produced the same degree of depolarization. When Na salts of anions less permeable than chloride (Br, I, NO₃) were used, the resting potentials in 116 mM solutions were close to those with chloride (±3mv). The depolarizations produced in low ionic strength solutions of these salts were significantly less than those with chloride.     

Diffusion in bile and its implications on detergency.

Diffusion coefficients of bile salts, lecithin, and cholesterol above the critical micelle concentration have been measured with the diaphragm cell at varying concentrations of bile salts, lecithin, and added electrolyte. The diffusion of the bile salt can be five times faster than that of the solubilized lipids. This is shown not to be an artifact of multicomponent diffusion, but a result of a different transport mechanism of the bile salt. As a consequence, the concentration of bile salt and lipids at the surface of a cholesterol gallstone can differ from those in the bile solution. The effects of this upon growth and dissolution in detergent solutions are discussed.     

A mechanistic analysis of the increase in the thermal stability of proteins in aqueous carboxylic acid salt solutions

The stability of proteins is known to be affected significantly in the presence of high concentration of salts and is highly pH dependent. Extensive studies have been carried out on the stability of proteins in the presence of simple electrolytes and evaluated in terms of preferential interactions and increase in the surface tension of the medium. We have carried out an in-depth study of the effects of a series of carboxylic acid salts: ethylene diamine tetra acetate, butane tetra carboxylate, propane tricarballylate, citrate, succinate, tartarate, malonate, and gluconate on the thermal stability of five different proteins that vary in their physico-chemical properties: RNase A, cytochrome c, trypsin inhibitor, myoglobin, and lysozyme. Surface tension measurements of aqueous solutions of the salts indicate an increase in the surface tension of the medium that is very strongly correlated with the increase in the thermal stability of proteins. There is also a linear correlation of the increase in thermal stability with the number of carboxylic groups in the salt. Thermal stability has been found to increase by as much as 22 C at 1 M concentration of salt. Such a high thermal stability at identical concentrations has not been reported before. The differences in the heat capacities of denaturation, deltaCp for RNase A, deduced from the transition curves obtained in the presence of varying concentrations of GdmCl and that of carboxylic acid salts as a function of pH, indicate that the nature of the solvent medium and its interactions with the two end states of the protein control the thermodynamics of protein denaturation. Among the physico-chemical properties of proteins, there seems to be an interplay of the hydrophobic and electrostatic interactions that lead to an overall stabilizing effect. Increase in surface free energy of the solvent medium upon addition of the carboxylic acid salts appears to be the dominant factor in governing the thermal stability of proteins.     

Brownian dynamics simulations of probe and self-diffusion in concentrated protein and DNA solutions.

We have developed a Brownian dynamics algorithm for simulating probe and self-diffusion in concentrated solutions of DNA and protein. In these simulations, proteins are represented as spheres with radii given by their hydrodynamic radii, while DNA is modeled as a wormlike chain of hydrodynamically equivalent spherical frictional elements. The molecular interaction potentials employed by the program allow for intramolecular stretching and bending motions of the DNA chains, short-range Lennard-Jones interactions, and long-range electrostatic interactions. To test the program, we have carried out simulations of bovine serum albumin (BSA) probe diffusion and DNA self-diffusion in solutions of short-chain DNA as a function of both DNA concentration and solution ionic strength. In addition, we report on simulations of BSA self-diffusion as a function of BSA concentration and ionic strength. Based on a comparison to available experimental data, we find that our simulations accurately predict these transport properties under conditions of physiological salt concentration and predict the stronger concentration dependence observed at lower salt concentrations. These results are discussed in light of the nature of the intermolecular interactions in such systems and the approximations and limitations of the simulation algorithm.     

Distribution of saccharides and salts on amphoteric ion-exchange resin

An amphoteric ion-exchange resin hardly shrank in 550 and 300 g/L glucose and sodium chloride solutions, respectively; however, the bed packed with a cation-exchange resin shrank considerably. From the distribution coefficients of some saccharides, the swelling pressure of the amphoteric ion-exchange resin was estimated to be 2.0 MPa at 25 °C. The distribution coefficients of glucose, galactose, fructose, and mannose were independent of their concentration and were about 0.621. On the other hand, the apparent distribution coefficients of NaF, NaCl, NaBr, NaI, LiCl, KCl, and CsCl largely depended on concentration. A model for the distribution of salts on the amphoteric resin was proposed, assuming an interaction between the anion of the salt and the positively charged fixed ions with binding constant B. The B values of the chloride salts were nearly the same (1.69–2.94 L/mol), while the values of the sodium salts were largely different depending on the anion.     

Calcium binding to bile salts

Calcium binding to bile salt monomers and micelles is an important issue with respect to the possible (but rare) precipitation of calcium bile salts in the gallbladder. In the present work the binding of Ca2+ to six bile salts was measured in solutions containing 2 to 100 mM bile salts by means of a calcium-sensitive dye, murexide, which determines the ionic calcium concentration. In solutions containing bile salt at concentration higher than 20 mM most, if not all, of the bound Ca2+ is associated with micellar surfaces. The results were analyzed by employing a model which combines specific binding with electrostatic equations and accounts for the system being a closed one. The analysis of Ca2+ binding data considered explicitly the presence of Na+ ions and yielded intrinsic binding coefficients for Ca2+ and Na+ which were utilized to explain and predict binding results for various concentrations of Ca2+, Na+ and bile salts. The calculations indicate that in saline solutions most of the surface sites were bound by Na+, whereas less than 10% were bound by Ca2+ even in the presence of 8 mM Ca2+. The binding of Ca2+ to bile salt micelles increases with pH. An increase in temperature results in reduced binding affinity of Ca2+ to the bile salt micelles.     

Sedimentation studies of DNA in concentrated solutions of magnesium salts

The sedimentation coefficient of calf thymus and of T7 DNA was determined at several concentrations up to saturation in solutions of each of the following salts: MgCl₂, MgBr₂, and MgSO₄. Under certain conditions, a plot of the product of the relative viscosity and sedimentation coefficient against the density of the solution has been found to be linear and to extrapolate to zero at a density corresponding to that of the solvated molecule. This behavior was realized in MgSO₄ solutions, the zero intercept occurring at a density of 1.41 g/cc, corresponding to a wafer activity of 0.89. The preferential solvation of DNA in MgSO₄ solutions calculated for this value is 10.5 moles water/mole of nucleotide, in good agreement with published values of solvation of DNA at the same water activity in univalent salt solutions. Since linear plots were not obtained in MgCl₂ and MgBr₂ solutions, buoyant densities could not be determined in these cases. The nonlinear behavior observed in MgCl₂ and MgBr₂ solutions may be due to a change in shape of the DNA molecule at the lower water activities reached in these solutions. The possibility of increased DNA–solute interactions in MgBr₂ and MgCl₂ solutions is also considered as an explanation for the difference in behavior between MgSO₄ and the two magnesium halides.     

Transfer free energies of peptide backbone unit from water to aqueous electrolyte solutions at 298.15 K

Transfer free energies (ΔG_tr) of amino acids from water to aqueous electrolyte solutions have been determined from the solubility measurements, as a function of salt concentration at 298.15 K under atmospheric pressure. The investigated aqueous systems contain amino acids of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly₂), triglycine (Gly₃), and tetraglycine (Gly₄) and cyclic glycylglycine (c(GG)) with an electrolyte compound of potassium chloride (KCl), potassium bromide (KBr) or potassium acetate (KAc). The solubilities of glycine and diglycine in aqueous solution decrease with increasing the concentration of salts (salting-out effect), whereas those of triglycine and tetraglycine increase with increasing the concentration of salts (salting-in effect). Furthermore, salting-in effect was found in aqueous c(GG)/KBr system, while salting-out effect was observed in aqueous c(GG)/KCl or c(GG)/KAc system. The experimental results were used to estimate the transfer free energies (Δg_tr) of the peptide backbone unit (–CH₂CONH–) from water to the aqueous electrolyte solutions. We developed a new trail to determine the activity coefficients (γ) for aqueous and aqueous electrolyte solutions using an activity coefficient model, with which the total contribution of transfer free energy between solute and the solvent was calculated. We compared the difference between neglecting and using the activity coefficients term in predicting ΔG_tr. Since the transfer free energy contribution is negative, interactions between the ionic salts and the peptide backbone unit of zwitterionic glycine peptides are favorable and thus the ionic salts destabilize these amino acids. It was also found that KBr stabilizes c(GG), whereas KCl and KAc destabilize c(GG). These results provide evidence for the existence of interactions between the amide unit and ionic salts, in aqueous solution, which may be of importance in maintaining protein structure as well as in protein–solute and protein–solvent interactions.