Extent of charge screening in aqueous polysaccharide solutions

The absorption optical system of a Beckman XL-I ultracentrifuge has been used to monitor the Donnan distribution of ions in polysaccharide solutions dialyzed against sodium phosphate buffer (pH 6.8, I 0.08) supplemented with 0.2 mM chromate as an indicator ion. For dextran sulfate, heparin, and polygalacturonate, the effective net charges are shown to be only one-third of those deduced from the chemical structures--a reflection of charge screening (counterion condensation) in aqueous polyelectrolyte solutions. Whereas the extent of charge screening for the first two polysaccharides agrees well with theoretical prediction, the disparity in the corresponding comparison for polygalacturonate reflects partial esterification of carboxyl groups, whereupon the experimental parameter refers to the effective charge per hexose residue rather than the effective fractional charge of each carboxyl group.     

Salting the charged surface: pH and salt dependence of protein G B1 stability

This study shows significant effects of protein surface charges on stability and these effects are not eliminated by salt screening. The stability for a variant of protein G B1 domain was studied in the pH-range of 1.5-11 at low, 0.15 M, and 2 M salt. The variant has three mutations, T2Q, N8D, and N37D, to guarantee an intact covalent chain at all pH values. The stability of the protein shows distinct pH dependence with the highest stability close to the isoelectric point. The stability is pH-dependent at all three NaCl concentrations, indicating that interactions involving charged residues are important at all three conditions. We find that 2 M salt stabilizes the protein at low pH (protein net charge is +6 and total number of charges is 6) but not at high pH (net charge is or=18). Furthermore, 0.15 M salt slightly decreases the stability of the protein over the pH range. The results show that a net charge of the protein is destabilizing and indicate that proteins contain charges for reasons other than improved stability. Salt seems to reduce the electrostatic contributions to stability under conditions with few total charges, but cannot eliminate electrostatic effects in highly charged systems.     

Allowance for effects of thermodynamic nonideality in sedimentation equilibrium distributions reflecting protein dimerization

This reexamination of a high-speed sedimentation equilibrium distribution for α-chymotrypsin under slightly acidic conditions (pH 4.1, I(M) 0.05) has provided experimental support for the adequacy of nearest-neighbor considerations in the allowance for effects of thermodynamic nonideality in the characterization of protein self-association over a moderate concentration range (up to 8 mg/mL). A widely held but previously untested notion about allowance for thermodynamic nonideality effects is thereby verified experimentally. However, it has also been shown that a greater obstacle to better characterization of protein self-association is likely to be the lack of a reliable estimate of monomer net charge, a parameter that has a far more profound effect on the magnitude of the measured equilibrium constant than any deficiency in current procedures for incorporating the effects of thermodynamic nonideality into the analysis of sedimentation equilibrium distributions reflecting reversible protein self-association.     

Studies of solute self-association by sedimentation equilibrium: allowance for effects of thermodynamic non-ideality beyond the consequences of nearest-neighbor interactions

A sedimentation equilibrium study of alpha-chymotrypsin self-association in acetate-chloride buffer, pH 4.1 I 0.05, has been used to illustrate determination of a dimerization constant under conditions where thermodynamic non-ideality is manifested beyond the consequences of nearest-neighbor interactions. Because the expressions for the experimentally determinable interaction parameters comprise a mixture of equilibrium constant and excluded volume terms, the assignment of reasonable magnitudes to the relevant virial coefficients describing non-associative cluster formation is essential for the evaluation of a reliable estimate of the dimerization constant. Determination of these excluded volume parameters by numerical integration over the potential-of-mean-force is shown to be preferable to their calculation by approximate analytical solutions of the integral for this relatively small enzyme monomer with high net charge (+10) under conditions of low ionic strength (0.05 M).     

Sulfate-binding protein dislikes protonated oxyacids. A molecular explanation

We have determined the effect of pH on the binding affinities of the conjugate bases of four different tetrahedral oxyacids to the sulfate-binding protein. The equilibrium dissociation constants of the binding of sulfate (Kd = 0.12 microM) and selenate (Kd = 5 microM) were found to be pH independent over the range pH 5 to pH 8.1, whereas chromate binding exhibited a pH dependence that is approximately attributable to the pK2 of the chromic acid. Phosphate was bound with an affinity five orders of magnitude weaker than that of sulfate. In light of the highly refined 2 A structure of the complex of the sulfate-binding protein with sulfate, and considering the protonation state and net charge of the various oxyacids, we conclude that the pH dependence of chromate binding and the extremely low affinity of phosphate are attributable mainly to a lack of hydrogen bond acceptors in the binding site. These studies demonstrate that the sulfate-binding site is stringently designed to bind tightly tetrahedral, fully ionized, oxyacid dianions. The presence of a donatable proton on the ligand reduces binding energy by approximately 7 kcal/mol.     

Effect of ion binding on protein transport through ultrafiltration membranes

Electrostatic interactions can have a significant impact on protein transmission through semipermeable membranes. Experimental data for the transport of bovine serum albumin (BSA) through a polyethersulfone ultrafiltration membrane were obtained in different salt solutions over a range of pH and salt concentrations. Net BSA charge under the same conditions was evaluated from mobility data measured by capillary electrophoresis. The results show that specific ionic composition, in addition to solution pH and ionic strength, can strongly affect the rate of protein transport through semipermeable ultrafiltration membranes. The effects of different ions on BSA sieving are due primarily to differences in ion binding to the protein, which leads to significant differences in the net protein charge at a given pH and ionic strength. This effect could be described in terms of an effective protein radius, which accounts for the electrostatic exclusion of the charged protein from the membrane pores. These results provide important insights into the nature of the electrostatic interactions in membrane systems.     

Effect of Metal Loading and Subcellular pH on Net Charge of Superoxide Dismutase-1

The net charge of a folded protein is hypothesized to influence myriad biochemical processes (e.g., protein misfolding, electron transfer, molecular recognition); however, few tools exist for measuring net charge and this elusive property remains undetermined—at any pH—for nearly all proteins. This study used lysine-acetyl “protein charge ladders” and capillary electrophoresis to measure the net charge of superoxide dismutase-1 (SOD1)—whose aggregation causes amyotrophic lateral sclerosis (ALS)—as a function of coordinated metal ions and pH. The net negative charge of apo-SOD1 was similar to predicted values; however, the binding of a single Zn^2 + or Cu^2 + ion reduced the net negative charge by a greater magnitude than predicted (i.e., ~ 4 units, instead of 2), whereas the SOD1 protein underwent charge regulation upon binding 2–4 metal ions. From pH5 to pH8 (i.e., a range consistent with the multiple subcellular loci of SOD1), the holo-SOD1 protein underwent smaller fluctuations in net negative charge than predicted (i.e., ~ 3 units, instead of ~ 14) and did not undergo charge inversion at its isoelectric point (pI = 5.3) but remained anionic. The regulation of SOD1 net charge along its pathways of metal binding, and across solvent pH, provides insight into its metal-induced maturation and enzymatic activity (which remains diffusion-limited across pH5–8). The anionic nature of holo-SOD1 across subcellular pH suggests that ~ 45 different ALS-linked mutations to SOD1 will reduce its net negative charge regardless of subcellular localization.     

Amyloid fibrillation in native and chemically-modified forms of carbonic anhydrase II: Role of surface hydrophobicity

Chemical modification or mutation of proteins may bring about significant changes in the net charge or surface hydrophobicity of a protein structure. Such events may be of major physiological significance and may provide important insights into the genetics of amyloid diseases. In the present study, fibrillation potential of native and chemically-modified forms of bovine carbonic anhydrase II (BCA II) were investigated. Initially, various denaturing conditions including low pH and high temperatures were tested to induce fibrillation. At a low pH of around 2.4, where the protein is totally dissociated, the apo form was found to take up a pre-molten globular (PMG) conformation with the capacity for fibril formation. Upon increasing the pH to around 3.6, a molten globular (MG) form became abundant, forming amorphous aggregates. Charge neutralization and enhancement of hydrophobicity by methylation, acetylation and propionylation of lysine residues appeared very effective in promoting fibrillation of both the apo and holo forms under native conditions, the rates and extents of which were directly proportional to surface hydrophobicity, and influenced by salt concentration and temperature. These modified structures underwent more pronounced fibrillation under native conditions, than the PMG intermediate form, observed under denaturing conditions. The nature of the fibrillation products obtained from intermediate and modified structures were characterized and compared and their possible cytotoxicity determined. Results are discussed in terms of the importance of surface net charge and hydrophobicity in controlling protein aggregation. A discussion on the physiological significance of the observations is also presented.     

Amyloid fibrillation in native and chemically-modified forms of carbonic anhydrase II: Role of surface hydrophobicity

Chemical modification or mutation of proteins may bring about significant changes in the net charge or surface hydrophobicity of a protein structure. Such events may be of major physiological significance and may provide important insights into the genetics of amyloid diseases. In the present study, fibrillation potential of native and chemically-modified forms of bovine carbonic anhydrase II (BCA II) were investigated. Initially, various denaturing conditions including low pH and high temperatures were tested to induce fibrillation. At a low pH of around 2.4, where the protein is totally dissociated, the apo form was found to take up a pre-molten globular (PMG) conformation with the capacity for fibril formation. Upon increasing the pH to around 3.6, a molten globular (MG) form became abundant, forming amorphous aggregates. Charge neutralization and enhancement of hydrophobicity by methylation, acetylation and propionylation of lysine residues appeared very effective in promoting fibrillation of both the apo and holo forms under native conditions, the rates and extents of which were directly proportional to surface hydrophobicity, and influenced by salt concentration and temperature. These modified structures underwent more pronounced fibrillation under native conditions, than the PMG intermediate form, observed under denaturing conditions. The nature of the fibrillation products obtained from intermediate and modified structures were characterized and compared and their possible cytotoxicity determined. Results are discussed in terms of the importance of surface net charge and hydrophobicity in controlling protein aggregation. A discussion on the physiological significance of the observations is also presented.     

Effects of net charge on the hydrogen-deuterium exchange parameters of lysozyme.

Possible effects of changes in net charge on protein hydrogen exchange rates were investigated by desalting hen egg-white lysozyme, which allowed its net charge to increase with decreasing pH in the acid region. Chloride ion-binding ratios, expressed as ratios of free to total Cl^?, were measured with a chloride-specific electrode at pH 5 on a 2.4% solution of a five-time-desalted product. This ratio was used to show a 97% reduction of the 11% Cl^? present in a commercial lysozyme preparation upon three passes of the enzyme through a column of ion-retardation resin. Net charges on the purified product were assigned from a combination of electrophoretic mobility and proton titration data gathered under minimal ionic strength conditions. The net charge on the desalted product increased by 1.64 units between pH 5.0 and 3.0. Hydrogendeuterium exchange studies on the purified lysozyme in D₂O were obtained using the near-infrared region of a Cary 14R spectrophotometer. The rate-pD profile for k₂, the rate constant for the intermediate class of exchanging hydrogens, showed a decrease in the apparent pD of minimum exchange rate of 0.3 units, when compared to that obtained earlier in 0.2 m added NaCl. However, the rate of exchange at pD minimum and the number of hydrogens in the class remained largely unaffected. A similar shift was observed for the rate-pD profile of the class 1 hydrogens. Thus, the effect of an increase in net positive charge is to shift the rate-pD profile to a lower pD. Moreover, the effect extended to the interior peptide hydrogens of this globular protein. Consequently, the exchange rates of all the observable hydrogens are altered by the net charge changes, and the effect appeared uniform. The shift can be accounted for quantitatively by applying electrostatic interaction terms to the acid and base catalytic constants characterizing the exchange process. The calculated electrostatic interaction factors in minimal salt and 0.2 m added NaCl were found to be 29 and 18% lower, respectively, than those obtained theoretically. Therefore, under conditions where changes in net charge may occur for a globular protein, the effect on hydrogen exchange rates can be estimated fairly well theoretically, especially at moderate ionic strengths.     

Cation-exchange high-performance liquid chromatography: separation of highly basic proteins using volatile acidic solvents

The chromatographic behavior of a number of globular proteins was studied on a Bio-Sil TSK CM-2-SW weak cation exchange HPLC column under acidic conditions. A linear gradient of 0-1 M NH4Ac in 1 M HOAc, inducing a convex pH gradient from 2.4-4.8, resulted in an excellent separation of highly basic proteins. For these proteins a linear relationship between isoelectric point and retention time was determined experimentally. The effect of pH and the ion composition of the eluting buffer system on this linear correlation was studied. Although the exact basis for protein separation on the CM-2-SW column at low pH is not clear yet, both the pH-dependent net positive charge per unit surface area and most likely the relative percentage of arginine in the total number of basic residues contribute to this separation. Because of the high resolving power and the high protein recovery obtained in a system using only acidic volatile buffer solutions, the cation exchanger is particularly suitable for the purification of nanogram amounts of acid-stable basic growth factors. The present sterile conditions (1 M HOAc/NH4Ac system, pH less than 4) and the easy removal of salt by lyophilization facilitate the detection of these proteins by biological assays.     

Chemical modification of lysine residues in lysozyme may dramatically influence its amyloid fibrillation

Studies on the aggregation of mutant proteins have provided new insights into the genetics of amyloid diseases and the role of the net charge of the protein on the rate, extent, and type of aggregate formation. In the present work, hen egg white lysozyme (HEWL) was employed as the model protein. Acetylation and (separately) citraconylation were employed to neutralize the charge on lysine residues. Acetylation of the lysine residues promoted amyloid formation, resulting in more pronounced fibrils and a dramatic decline in the nucleation time. In contrast, citraconylation produced the opposite effect. In both cases, native secondary and tertiary structures appeared to be retained. Studies on the effect of pH on aggregation suggested greater possibilities for amorphous aggregate formation rather than fibrillation at pH values closer to neutrality, in which the protein is known to take up a conformation more similar to its native form. This is in accord with reports in the literature suggesting that formation of amorphous aggregates is more favored under relatively more native conditions. pH 5 provided a critical environment in which a mixture of amorphous and fibrillar structures were observed. Use of Tango and Aggrescan software which describe aggregation tendencies of different parts of a protein structure suggested critical importance of some of the lysine residues in the aggregation process. Results are discussed in terms of the importance of the net charge in control of protein–protein interactions leading to aggregate formation and possible specific roles of lysine residues 96 and 97.     

Glycocalyx electrostatic potential profile analysis: ion, pH, steric, and charge effects

The Poisson-Boltzmann equation is modified to consider charge ionogenicity, steric exclusion, and charge distribution in order to describe the perimembranous electrostatic potential profile in a manner consistent with the known morphology and biochemical composition of the cell's glycocalyx. Exact numerical and approximate analytical solutions are given for various charge distributions and for an extended form of the Donnan potential model. The interrelated effects of ionic conditions, bulk pH, ion binding, local dielectric, steric volume exclusion, and charge distribution on the local potential, pH, and charge density within the glycocalyx are examined. Local charge-induced, potential-mediated pH reductions cause glycocalyx charge neutralization. Under certain conditions, local potentials may be insensitive to ionic strength or may decrease in spite of increasing charge density. The volume exclusion of the glycocalyx reduces the local ion concentration, thereby increasing the local potential. With neutral lipid membranes, the Donnan and surface potential agree if the glycocalyx charge distribution is both uniform and several times thicker than the Debye length (approximately 20 A in thickness under physiological conditions). Model limitations in terms of application to microdomains or protein endo- and ectodomains are discussed.     

Ion exchange chromatography of antibody fragments

Effects of pH and conductivity on the ion exchange chromatographic purification of an antigen-binding antibody fragment (Fab) of pI 8.0 were investigated. Normal sulfopropyl (SP) group modified agarose particles (SP Sepharosetrade mark Fast Flow) and dextran modified particles (SP Sepharose XL) were studied. Chromatographic measurements including adsorption isotherms and dynamic breakthrough binding capacities, were complemented with laser scanning confocal microscopy. As expected static equilibrium and dynamic binding capacities were generally reduced by increasing mobile phase conductivity (1-25 mS/cm). However at pH 4 on SP Sepharose XL, Fab dynamic binding capacity increased from 130 to 160 (mg/mL media) as mobile phase conductivity changed from 1 to 5 mS/cm. Decreasing protein net charge by increasing pH from 4 to 5 at 1.3 mS/cm caused dynamic binding capacity to increase from 130 to 180 mg/mL. Confocal scanning laser microscopy studies indicate such increases were due to faster intra-particle mass transport and hence greater utilization of the media's available binding capacity. Such results are in agreement with recent studies related to ion exchange of whole antibody molecules under similar conditions.     

Specific ion effects: why the properties of lysozyme in salt solutions follow a Hofmeister series

Protein solubility in aqueous solutions depends in a complicated and not well understood way on pH, salt type, and salt concentration. Why for instance does the use of two different monovalent salts, potassium thiocyanate and potassium chloride, produce such different results? One important and previously neglected source of ion specificity is the ionic dispersion potential that acts between each ion and the protein. This attractive potential is found to be much stronger for SCN(-) than it is for Cl(-). We present model calculations, performed within a modified ion-specific double-layer theory, that demonstrate the large effect of including these ionic dispersion potentials. The results are consistent with experiments performed on hen egg-white lysozymes and on neutral black lipid membranes. The calculated surface pH and net lysozyme charge depend strongly on the choice of anion. We demonstrate that the lysozyme net charge is larger, and the corresponding Debye length shorter, in a thiocyanate salt solution than in a chloride salt solution. Recent experiments have suggested that pK(a) values of histidines depend on salt concentration and on ionic species. We finally demonstrate that once ionic dispersion potentials are included in the theory these results can quantitatively be reinterpreted in terms of a highly specific surface pH (and a salt-independent pK(a)).     

Probing the mechanism of amyloidogenesis through a tandem repeat of the PI3-SH3 domain suggests a generic model for protein aggregation and fibril formation

Aggregation of the SH3 domain of the PI3 kinase, both as a single domain and as a tandem repeat in which the C terminus of one domain is linked to the N terminus of another by a flexible linker of ten glycine/serine residues, has been studied under a range of conditions in order to investigate the mechanism of protein aggregation and amyloid formation. The tandem repeat was found to form amyloid fibrils much more readily than the single domain under the acidic conditions used here, and the fibrils themselves have higher morphological homogeneity. The folding-unfolding transition of the PI3-SH3 domain shows two-state behaviour and is pH dependent; at pH 3.6, which is near the pH mid-point for folding and only slightly below the isoelectric point of the protein, both the single domain and the tandem repeat spontaneously form broad distributions of soluble oligomers without requirement for nucleation. Under prolonged incubation under these conditions, the oligomers convert into thin, curly fibrils that interact with thioflavin-T, suggesting that they contain an organised beta-sheet structure. Under more acidic conditions (pH 2.0) where the proteins are fully denatured and carry a positive net charge, long, straight fibrils are formed in a process having a pronounced lag phase. The latter was found to be reduced dramatically by the addition of oligomers exceeding a critical size of approximately 20 molecules. The results suggest that the process of aggregation of these SH3 domains can take place by a variety of mechanisms, ranging from downhill formation of relatively amorphous species to nucleated formation of highly organised structures, the relative importance of which varies greatly with solution conditions. Comparison with the behaviour of other amyloidogenic systems suggests that the general mechanistic features outlined here are likely to be common to at least a wide variety of peptides and proteins.     

Determining the net flux of charge released by maize roots by directly measuring variations of the alkalinity in the nutrient solution

The net flux of charge released by maize, i.e. the strong ion exchange balance between the roots and their environment, was determined in acidic and alkaline solutions, i.e. solutions with a low and a high pH buffering capacity, respectively. The work was based on direct measurement of total alkalinity in culture solutions over a period of several days.The results show there was little difference in the net flux of charge released by maize in acidic and alkaline solutions: In both cases, approximately –1 mol_c (kg DM)^–1 s^–1. As the maize was grown in a non-limiting nitrate solution, the charge flux was negative, corresponding to a net release of hydroxyls into the rhizosphere. In contrast, the change in the amounts of free protons in the solution was approximately 1 nmol (kg DM)^–1 s^–1, i.e. 3 orders of magnitude lower than the net charge flux. Moreover, it was negative in acidic media , i.e. the solution pH increased, and positive in alkaline media, i.e. the solution pH decreased. This decrease probably resulted from the release of inorganic carbon by the roots. The effect on the change in solution pH was only slight in acidic conditions but considerable in alkaline conditions, where it reduced the pH even though the culture solution was alkalinised by the roots.The difference in the way that acidic and alkaline solutions function demonstrates the importance of the pH buffering capacity of the solution in determining the net flux of charge released by the plants. It underlines the difficulty of estimating the net charge flux from pH change measurements in the rhizosphere.     

Emulsion-stabilizing properties of chitosan in the presence of whey protein isolate: Effect of the mixture ratio, ionic strength and pH

The emulsion-stabilizing properties of a chitosan preparation were compared as a function of the whey protein isolate/chitosan mixture ratio (WPI/CNI) and the ionic strength (μ), at pH 5.5 and 6.0. At both pH conditions, general decreases in emulsion stability towards charge neutralization flocculation and syneresis were observed at WPI/CNI > 5. This was particularly evident at pH 6.0, due to a lower surface net charge (lower electrostatic stabilization). In counterpart, when μ was increased, the higher load of chitosan at pH 6.0 produced higher stabilities (higher steric stabilization), in spite of comparable decreases of surface net charge at both pH conditions. The transition from soluble to insoluble protein–chitosan complex formation in mixtures at pH 6.0 and WPI/CNI > 5.0 was due to an emulsion destabilization towards syneresis, whereas soluble complex formation at pH 5.5 also produced syneresis. It showed that soluble protein–chitosan adsorbing complex formation prior homogenization is not essentially linked to emulsion stabilization.     

Effect of Hofmeister ions on protein thermal stability: roles of ion hydration and peptide groups?

We have systematically explored the Hofmeister effects of cations and anions (0.3-1.75 M range) for acidic Desulfovibrio desulfuricans apoflavodoxin (net charge −19, pH 7) and basic horse heart cytochrome c (net charge +17, pH 4.5). The Hofmeister effect of the ions on protein thermal stability was assessed by the parameter dT_trs/d[ion] (T_trs; thermal midpoint). We show that dT_trs/d[ion] correlates with ion partition coefficients between surface and bulk water and ion surface tension effects: this suggests direct interactions between ions and proteins. Surprisingly, the stability effects of the different ions on the two model proteins are similar, implying a major role of the peptide backbone, instead of charged groups, in mediation of the interactions. Upon assessing chemical/physical properties of the ions responsible for the Hofmeister effects on protein stability, ion charge density was identified as most important. Taken together, our study suggests key roles for ion hydration and the peptide group in facilitating interactions between Hofmeister ions and proteins.     

Analysis of the thermodynamic non-ideality of proteins by sedimentation equilibrium experiments

This paper presents a modified method to determine experimentally the second virial coefficient of protein solutions by sedimentation equilibrium experiments. The improvement is based on the possibility of fitting simultaneously up to seven radial concentration distribution curves of solutions with different loading concentrations. The possibility of precise determination of the second virial coefficient allows estimation of the net charge and the excluded volume of a monomeric protein. Application of the method is demonstrated for lysozyme and ovalbumin. In 0.1 M sodium acetate buffer, pH 4.5, the second virial coefficient of hen egg white lysozyme amounts to 24 +/- 1 ml/g. Analysis based on spherical particle theory yield an excluded volume of 3.5 ml/g and a charge dependent value of 20.5 ml/g which is induced by a net charge number of 14.1 +/- 1. Under low salt conditions self-association processes on lysozyme are unfavorable due to electrostatic repulsion. To overcome these repulsive contributions, either a shift to neutral pH or addition of at least 2% NaCl is necessary. In this way the charge dependent contribution decreases below the value responsible for the excluded volume and allows crystallization of the protein. Similar effects can be observed with ovalbumin. The high virial coefficient observed at pH 8.5 is induced by the high net charge number of 27 +/- 1.