Influence of pH value and salts on the adsorption of lysozyme in mixed‐mode chromatography

Mixed‐mode chromatography (MMC) is an interesting technique for challenging protein separation processes which typically combines adsorption mechanisms of ion exchange (IEC) and hydrophobic interaction chromatography (HIC). Adsorption equilibria in MMC depend on multiple parameters but systematic studies on their influence are scarce. In the present work, the influence of the pH value and ionic strengths up to 3000 mM of four technically relevant salts (sodium chloride, sodium sulfate, ammonium chloride, and ammonium sulfate) on the lysozyme adsorption on the mixed‐mode resin Toyopearl MX‐Trp‐650M was studied systematically at 25℃. Equilibrium adsorption isotherms at pH 5.0 and 6.0 were measured and compared to experimental data at pH 7.0 from previous work. For all pH values, an exponential decay of the lysozyme loading with increasing ionic strength was observed. The influence of the pH value was found to depend significantly on the ionic strength with the strongest influence at low ionic strengths where increasing pH values lead to decreasing lysozyme loadings. Furthermore, a mathematical model that describes the influence of salts and the pH value on the adsorption of lysozyme in MMC is presented. The model enables predicting adsorption isotherms of lysozyme on Toyopearl MX‐Trp‐650M for a broad range of technically relevant conditions.     

Further studies on the contribution of electrostatic and hydrophobic interactions to protein adsorption on dye-ligand adsorbents.

The adsorption equilibria of bovine serum albumin (BSA), gamma-globulin, and lysozyme to three kinds of Cibacron blue 3GA (CB)-modified agarose gels, 6% agarose gel-coated steel heads (6AS), Sepharose CL-6B, and a home-made 4% agarose gel (4AB), were studied. We show that ionic strength has irregular effects on BSA adsorption to the CB-modified affinity gels by affecting the interactions between the negatively charged protein and CB as well as CB and the support matrix. At low salt concentrations, the increase in ionic strength decreases the electrostatic repulsion between negatively charged BSA and the negatively charged gel surfaces, thus resulting in the increase of BSA adsorption. This tendency depends on the pore size of the solid matrix, CB coupling density, and the net negative charges of proteins (or aqueous - phase pH value). Sepharose gel has larger average pore size, so the electrostatic repulsion-effected protein exclusion from the small gel pores is observed only for the affinity adsorbent with high CB coupling density (15.4 micromol/mL) at very low ionic strength (NaCl concentration below 0.05 M in 10 mM Tris-HCl buffer, pH 7.5). However, because CB-6AS and CB-4AB have a smaller pore size, the electrostatic exclusion effect can be found at NaCl concentrations of up to 0.2 M. The electrostatic exclusion effect is even found for CB-6AS with a CB density as low as 2.38 micromol/mL. Moreover, the electrostatic exclusion effect decreases with decreasing aqueous-phase pH due to the decrease of the net negative charges of the protein. For gamma-globulin and lysozyme with higher isoelectric points than BSA, the electrostatic exclusion effect is not observed. At higher ionic strength, protein adsorption to the CB-modified adsorbents decreases with increasing ionic strength. It is concluded that the hydrophobic interaction between CB molecules and the support matrix increases with increasing ionic strength, leading to the decrease of ligand density accessible to proteins, and then the decrease of protein adsorption. Thus, due to the hybrid effect of electrostatic and hydrophobic interactions, in most cases studied there exists a salt concentration to maximize BSA adsorption.     

The Influence of pH and Ionic Strength on the Electrokinetic Stability of the Human Erythrocyte Membrane

The electrokinetic stability of washed normal human erythrocytes is discussed from the point of view of pH, ionic strength, and composition of the suspending medium. Many of the electrophoretic characteristics at low ionic strengths (sorbitol to maintain the tonicity), such as the isopotential points, are shown to arise principally from adsorption of hemolysate. The concept of electrokinetically stable, metastable, and unstable states for the red cell at various ionic strengths is introduced in preference to the general term "cell injury." In the stable state which exists around pH 7.4 for ionic strengths >0.007, no adsorption of hemolysate occurs, in the metastable state reversible adsorption of hemolysate occurs, and in the unstable state, in which ionic strengths and pH ranges are outside the metastable range, the membrane undergoes irreversible hemolysate adsorption or more general hydrolytic degradation. It is deduced from the equivalent binding of CNS, I, Cl, and F, the pH mobility relationships, and the conformation of the ionic strength data in the stable state to a Langmuir adsorption isotherm, that the membrane of the human erythrocyte behaves as a macropolyanion whose properties are modified by gegen ion association and in some instances by hemolysate adsorption. The experimental results are insufficient to establish conclusively the nature of the ionogenic groupings present in the membrane interphase.     

Adsorption of gamma-globulin, a model protein for antibody, on colloidal particles

The effect of surface properties on the adsorption of bovine gamma-globulin, a model protein for antibody, was studied. Polystyrene latex (PS), hydrophilic copolymer lattices of styrene/2-hydroxyethyl methacrylate [P(S/HEMA)], styrene/ methacrylic acid [P(S/MAA)] and methyl methacrylate/ 2-hydroxyethyl methacrylate [P(MMA/HEMA)], and colloidal silica were used. The adsorption isotherms of gamma-globulin on these colloidal particles were measured as a function of pH and ionic strength. The hydrophilic particles showed low affinities for gamma-globulin at alkaline pH, while PS showed high affinities for gamma-globulin over the whole range of pH and ionic strength. The gamma-globulin adsorption on hydrophilic particles was highly reversible with respect to the pH and ionic strength compared with that on PS. These differences indicate that the dominant driving forces of adsorption are related to the hydrophilicity of particles. The adsorption isotherms of all colloidal particles showed the plateau values, and the order of maximum values of plateau adsorption was P(S/MAA) > PS or P(S/HEMA), silica > P(MMA/HEMA). Thus, they were also affected by the charged groups and the hydrophilicity of the surfaces. On the other hand, the plateau values of all colloidal particles were more or less symmetrical with a maximum at around the isoelectric point of gamma-globulin at an ionic strength of 0.01. This behavior is attributed to the important role of the lateral interaction between the adsorbed molecules at low ionic strength.     

Binding of bromophenol blue to bovine serum albumin and its succinylated forms

Interaction of bromophenol blue with bovine serum albumin and its five succinylated forms was studied spectrophotometrically at three different ionic strengths, i.e. 0.04, 0.15 and 1.0 and at two different pH values, namely pH 7.0 and pH 5.0 respectively. Results showed a decrease in bromophenol blue binding on increasing succinylation at low ionic strengths. This decrease was more marked at pH 7.0 than pH 5.0. However, at both the pH values binding returned to a significant degree on increasing the ionic strength to 1.0. Succinylation also caused marked conformational changes at pH 7.0 and ionic strength 0.15 as evidenced by changes in hydrodynamic properties and reduction in antigen-antibody precipitin reaction. However, an increase in ionic strength to 1.0 or decrease in pH to 5.0 caused significant reversal in hydrodynamic parameters. These studies show that lysine residues of bovine serum albumin are not important in bromophenol blue binding.     

Hydrogen-ion titration curve of ovomucoid.

A systematic study of the H+ titration curve of purified ovomucoid was made at three temperatures (15, 25 and 35 degrees C) and three ionic strengths (0.05, 0.15 and 1.0). In all, 49 protons were dissociated reversibly in the pH range, 2.0-12.0. From the analysis of the results up to pH 12.0, the numbers of different dissociable groups per 28 300 g protein, together with their intrinsic pK values in parentheses were found tp be' 27 sode-chain carboxyl (pKint=4.0), four imidazole (pKint=6.5), one alpha-amino (pKint=7.5), 12 epsilon-amino (pKint=9.6), one guanidino (pKint=11.8) and one alpha-carboxyl group with abnormally low pK. The total number of basic nitrogens per mole of the protein was 22 so that four guanidino groups remained untitrated up to pH 12.0. Spectrophotometric titration showed that three out of five phenolic groups were titrated reversibly up to pH 11.9 with an intrinsic pK of 10.25; the remaining two groups became accessible only on protein denaturation. Viscosity results suggested absence of conformational change in the pH range 2.0-11.2. This explains the constancy of the pK values of carboxyl groups in the pH range 2.0-5.0. The empirical value of the electrostatic interaction factor, w, was 0.04, both in the carboxyl and phenolic regions.     

Protein adsorption at solid-liquid interfaces: Part I--Affinities of proteins for alumina surface

Extent of adsorption (gamma pw) of bovine serum albumin, beta-lactoglobulin, gelatin and myosin at the alumina-water interface has been measured as function of protein concentration (Cp) at several temperatures, pH, and ionic strengths of the medium. gamma pw for proteins in most cases increases with increase of protein concentration but it attains maximum value gamma pw(m) when Cp is high. Values of maximum adsorption have been examined in terms of molecular orientation, molecular size and shape and unfolding of the packed proteins at the interface. In few cases, gamma pw increases with increase of Cp without reaching a real state of saturation as a result of aggregation of molecules or extensive unfolding of the protein at the interface. In the case of beta-lactoglobulin at pH 5.2 and ionic strength 0.05, gamma pw in high concentration region decreases to zero value when Cp increases. For myosin at 45 degrees C and pH 6.4, and also at 27 degrees and pH 7.8, the values of gamma pw are all negative and these negative values increase with increase of Cp. All these results have been explained in terms of significant competitions of water and protein for binding to the surface sites of the powdered alumina. Adsorption of myosin has also been found to be affected in the presence of NaCl, KCl, CaCl2, KI, Na2SO4, LiCl and urea. The relative affinities of the adsorption of various proteins for the surface of alumina at different physical conditions of the system have been compared in terms of maximum values of adsorption attained when gamma pw is varied with Cp. The affinities are shown to be compared more precisely in terms of the standard free energy decrease for the saturation of the surface by protein as a result of the change in its concentration from zero to unity in the mole fraction scale.     

Adsorption of biopolymers at hydrophilic cellulose-water interface

The extent of adsorption (Gamma2(1)) of bovine serum albumin (BSA), beta-lactoglobulin, lysozyme, gelatin, and DNA from aqueous solution onto the hydrophilic surface of cellulose has been measured as function of biopolymer concentration at different temperatures, pHs, and ionic strengths, and in the presence of a high concentration of inorganic salts and denaturants. In all cases, the value of Gamma2(1) increases with the increase of biopolymer concentration (X2) in bulk and it attains a maximum value at a critical mole fraction concentration X2m. The value of Gamma2m depends upon the nature of protein, temperature, pH, and ionic strength, as well as the nature of neutral salts present in excess. Gamma2m for proteins at a fixed physicochemical condition stands in the following order: Gelatin>betalactoglobulin>lysozyme>BSA. The isotherms for adsorption of DNA nucleotides on cellulose surface at pH 4.0 have been compared at different temperatures and ionic strengths, and in the presence of high concentration of inorganic salts LiCl, NaCl, KCl, and Na2SO4. Values of Gamma2m for different systems have been evaluated and critically compared. At pH 6.0 and 8.0, Gamma2(1) values of DNA nucleotides on cellulose are all negative due to the excess positive hydration of cellulose. At pH 4.0, adsorption of nucleotides of acid, alkali, and heat-denatured DNA widely differ from each other and in the presence of excess concentration of urea becomes negative. The probable mechanisms of biopolymer-cellulose adsorption in terms of polymer hydration, steric interaction, London-van der Waals, hydrophobic, and other types of interactions have been discussed qualitatively. The standard free energy change for the adsorption of protein and DNA nucleotides on the cellulose surface at the state of adsorption saturation has been calculated in kJ per kg of cellulose using an integrated form of the Gibbs adsorption equation. The relation between DeltaG degrees and maximum affinities between biopolymers and the polysaccharide interface have been discussed for various systems.     

Temperature- and ionic strength-induced conformational changes in the lipid head group region of liposomes as suggested by zeta potential data.

Neutral liposomes composed of DMPC (dimyristoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine) or DSPC (distearoylphosphatidylcholine) are found to exhibit non-zero zeta potentials in an electric field even when they are dispersed in solution at pH 7.4. A model for the orientation of lipid head groups is proposed to explain the observed non-zero zeta potentials. The dependence of the zeta potential on temperature and ionic strength is analyzed via this model to obtain the information on the direction of the lipid head group in the liposome surface region. The direction of the lipid head group is found to be sensitive to the temperature and the ionic strength of the medium. At low ionic strengths, the phosphatidyl groups are located at the outer portion of the head group region. At constant temperature, as the ionic strength increases, the choline group approaches the outer region of the bilayer surface while the phosphatidyl group hides behind the surface. At the phase transition temperature of the lipid, the phosphatidyl group lies in the outer-most region of the surface and the choline group is in the inner-most region.     

Cadmium adsorption by an organic soil: a comparison of some humic – metal complexation models

Abstract

The retention of Cd by an organic soil was investigated as a function of pH and ionic strength. The adsorption of Cd at pH values from 2 to 11 at two ionic strengths (0.053 M and 0.017 M LiNO₃) were found to be a function of both pH and ionic strength. Four Cd-humic complexation models were evaluated in order to test the applicability of these models to fit data from batch adsorption experiments. The models varied greatly in their complexity and implicit assumptions. Three were discrete functional group models – a simple diprotic acid model, a two diprotic acid model and the Windermere Humic Aqueous Acid (WHAM) model, and a continuous functional group model - the non-ideal competitive adsorption (NICA) model. The concentration of proton binding sites in the soil was found to be 4.51 mol kg^-1. The NICA and WHAM models were more successful than either a simple diprotic acid model or a two diprotic acid model at modeling Cd complexation by the organic soil, although both underestimated adsorption at very high pH values.     

Boron adsorption by maize cell walls

Boron adsorption by cell walls isolated from corn (Zea mays) roots was investigated as a function of solution pH and ionic strength. Adsorption increased with increasing solution pH from pH 4.5 to 10, exhibited an adsorption maximum at pH 10–10.5, and decreased with increases in pH above 10.5. Boron adsorption increased with increasing solution ionic strength indicating the formation of strong inner-sphere surface complexes. A surface complexation model, the constant capacitance model was well able to describe the B adsorption data, optimizing two B surface complexes and the dissociation constant for the surface functional group, XOH. The large absolute value of the dissociation constant is consistent with phenolic functional groups.     

Adsorption of bovine hemoglobin onto spherical polyelectrolyte brushes monitored by small-angle X-ray scattering and Fourier transform infrared spectroscopy

The adsorption of bovine hemoglobin (BHb) onto colloidal spherical polyelectrolyte brushes (SPBs) is studied by a combination of small-angle X-ray scattering (SAXS) and Fourier transform infrared spectroscopy (FTIR). The SPBs consist of a polystyrene core onto which long chains of poly(styrene sulfonic acid) are grafted. Hemoglobin is a tetrameric protein that disassembles at low pH's and high ionic strengths. The protein is embedded into the brush layer composed of strong polyacids. Thus, the protein is subjected to a pH and ionic strength that largely differs from the bulk solution. At low ionic strengths up to 650 mg of BHb per gram of SPB could be immobilized. The analysis of the particles loaded with protein by SAXS demonstrates that the protein enters deeply into the brush. A large fraction of hemoglobin is bound at the surface of the polystyrene core. We attribute this strong affinity to hydrophobic interactions between the protein and the polystyrene core. The other protein molecules are closely correlated with the polyelectrolyte chains. The secondary structure of the protein within the brush was studied by FTIR spectroscopy. The analysis revealed a significant disturbance of the secondary structure of the tetrameric protein. The content of alpha-helix is significantly lowered compared to the native conformation. Moreover, there is an increase of beta-sheet structure as compared to the native conformation. The partial loss of the structural integrity of the hydrophobic protein is due to hydrophobic interactions with the hydrophobic polystyrene core. Hydrophobic interactions with the phenyl groups of the poly(styrene sulfonate) chains influence the secondary structure as well. These findings indicate that changes of the secondary structure play a role in the uptake of hemoglobin into the poly(styrene sulfonate) brushes.     

Influence of pH and ionic strength on the adsorption, leaching and activity of myoglobin immobilized onto ordered mesoporous silicates

Myoglobin has been immobilized onto different ordered mesoporous silicates. The effect of the pH on the adsorption, leaching and activity was studied. The results showed that the maximum amount of protein was adsorbed at a pH 6.5, just below the protein isoelectric point (7–7.3). There was no effect of increasing ionic strength on the adsorption profile at different pH values. The adsorption is rationalized in terms of local electrostatic forces acting between the enzyme and the silica surface as well as hydrophobic interactions close to the protein isoelectric point, whereas at low pH the global charges give rise to protein–protein repulsion and at high pH enzyme–silica repulsion. Higher amounts of immobilized myoglobin were leached at a pH 4, while lower amounts were leached at pH 6.5. The catalytic activity of myoglobin immobilized onto SBA-15 showed optimal activity at a pH 6.5 in comparison to a pH of 5 for the free form.     

SURFACE CHARGE OF CHOLINE ACETYLTRANSFERASE FROM DIFFERENT SPECIES

—The adsorption of partially purified choline acetyltransferase (ChAc) from cat, rat, guinea-pig and pigeon brains by the cation exchange resins, CM-Sephadex (C-50) and Amberlite CG-50 II, was studied at various pH values and ionic strengths. ChAc from cat and rat were more strongly adsorbed by cation exchangers and therefore have a stronger net positive surface charge than those from guinea pig and pigeon. Experiments showed that the difference in adsorption between these two groups of enzymes could not be explained by overloading of the resin, by competitive effect of other proteins present in the enzyme preparations or by the presence of any component suppressing the adsorption of ChAc in any of the enzyme preparations. The adsorption of ChAc by a cation exchanger is very similar to its binding to synaptosome membranes. The significance of the positive surface charge of ChAc in studies on the compartmentation of ChAc in synaptosomes is discussed.     

No salting-in of lysozyme chloride observed at low ionic strength over a large range of pH.

Solubility of lysozyme chloride was determined in the absence of added salt and in the presence of 0.05-1.2 M NaCl, starting from isoionic lysozyme, which was then brought to pH values from 9 to 3 by addition of HCl. The main observation is the absence of a salting-in region whatever the pH studied. This is explained by a predominant electrostatic screening of the positively charged protein and/or by adsorption of chloride ions by the protein. The solubility increases with the protein net charge at low ionic strength, but the reverse is observed at high ionic strength. The solubility of lysozyme chloride seems to become independent of ionic strength at pH approximately 9.5, which is interpreted as a shift of the isoionic pH (10.8) to an isoelectric pH due to chloride binding. The crystallization at very low ionic strength, where lysozyme crystallizes at supersaturation values as low as 1.1, amplifies the effect of pH on protein solubility. Understanding the effect of the net charge and of ionic strength on protein-protein interactions is valuable not only for protein crystal growth but more generally also for the formation of protein-protein or protein-ligand complexes.     

Mobility of adsorbed proteins: a Brownian dynamics study

We simulate the adsorption of lysozyme on a solid surface, using Brownian dynamics simulations. A protein molecule is represented as a uniformly charged sphere and interacts with other molecules through screened Coulombic and double-layer forces. The simulation starts from an empty surface and attempts are made to introduce additional proteins at a fixed time interval that is inversely proportional to the bulk protein concentration. We examine the effect of ionic strength and bulk protein concentration on the adsorption kinetics over a range of surface coverages. The structure of the adsorbed layer is examined through snapshots of the configurations and quantitatively with the radial distribution function. We extract the surface diffusion coefficient from the mean square displacement. At high ionic strengths the Coulombic interaction is effectively shielded, leading to increased surface coverage. This effect is quantified with an effective particle radius. Clustering of the adsorbed molecules is promoted by high ionic strength and low bulk concentrations. We find that lateral protein mobility decreases with increasing surface coverage. The observed trends are consistent with previous theoretical and experimental studies.     

Protein adsorption at solid-liquid interfaces: Part III--Adsorption from ternary protein mixture.

Simultaneous adsorption of bovine serum albumin (BSA), beta-lactoglobulin and gelatin from aqueous solutions of their ternary mixture to the alumina-water interface has been studied as a function of protein concentration at different values of pH, ionic strength, temperature and weight fraction ratios of proteins. At a fixed weight fraction of beta-lactoglobulin, preferential adsorption (gamma w(lac)) of this protein significantly depends on the amounts of BSA and gelatin present in the solution before adsorption. At higher ranges of protein concentrations, extent of adsorption (gamma w(ser)) of BSA decreases sharply with increase of gamma w(lac) until gamma w(ser) becomes significantly negative, thereby indicating that beta-lactoglobulin and water preferentially adsorbed at the interface are responsible for complete displacement of BSA from the surface. On the other hand, adsorption (gamma w(gel)) of gelatin under similar situation increases mutually with increase in the values of gamma w(lac) in many systems. In few systems, gamma w(gel) also decreases with increase of gamma w(lac) depending upon solution parameters. At pH 5.2, increase of ionic strength and temperature, respectively, increases the extent of adsorption of each protein in the mixture considerably. Extents of adsorption of all proteins are observed to increase when pH is changed from 5.2 to 6.4. The affinities of different proteins in the mixture are expressed in unified scales either in terms of maximum extents of total adsorption or in terms of standard free energies of adsorption of protein mixtures with respect to surface saturation.     

Accumulation of zinc and cadmium by Cytophaga johnsonae

Passive and active accumulation of zinc and cadmium by a common soil and freshwater bacterium, Cytophaga johnsonae, was studied using a radio-tracer batch distribution technique. The effects of variation of pH (3–10), as well as of ionic strength (0.007 and 0.07 m) on passive accumulation of the metals were examined. For both zinc and cadmium, accumulation was mainly due to passive processes, such as surface adsorption and/or diffusion into the periplasm. However, at low zinc concentrations, accumulation increased when glucose was added, suggesting an active uptake; at higher zinc concentrations such uptake was not detected, probably because it was masked by the stronger sorption properties of the cell wall. Adsorption of the metals was pH dependent: at higher ionic strength, accumulation was enhanced at pH values above 7; at lower ionic strength, adsorption differed and was markedly higher, with increased accumulation between pH 5 and 8.     

The influence of ionic strength, pH and a protein layer on the interaction between Streptococcus mutans and glass surfaces

The initial interaction between Streptococcus mutans and hard surfaces has been investigated using a rotating disc technique. The deposition to clean and BSA-coated glass of two strains of S. mutans, FA-1 (serotype b) and KPSK2 (serotype c), which exhibit different surface properties, was studied. Organisms were harvested from cultures grown in a chemostat at a dilution rate of 0.06 h-1 and suspended in NaCl solutions of defined ionic strengths and pH values. The deposition of both strains showed a strong dependence on electrolyte concentration, particularly at low ionic strengths, which was inversely related to the zeta potentials of the organisms. Similarly, the ionic strength at which maximum deposition was first noted (critical coagulation concentration) for the two strains correlated with their relative potentials. Deposition was insensitive to changes in pH at an electrolyte concentration of 0.05 M. The maximum observed deposition did not approach values predicted by theory, suggesting that a further barrier to deposition, other than electrostatic repulsion, might exist. Under all experimental conditions, some of the deposited bacteria were observed to be oscillating, suggesting that they were held at a distance from the collector surface. The cells did not, however, appear to be deposited in a secondary minimum predicted by DLVO theory hence it may be that long-range polymer interactions are also involved in the deposition of these organisms.     

Differential reactivity of maleimide and bromoacetyl functions with thiols: application to the preparation of liposomal diepitope constructs

The comparative reactivity of maleimide and bromoacetyl groups with thiols (2-mercaptoethanol, free cysteine, and cysteine residues present at the N-terminus of peptides) was investigated in aqueous media. These studies were performed (i) with water-soluble functionalized model molecules, i.e., polyoxyethylene-based spacer arms that could also be coupled to lipophilic anchors destined to be incorporated into liposomes, and (ii) with small unilamellar liposomes carrying at their surface these thiol-reactive functions. Our results indicate that an important kinetic discrimination (2-3 orders of magnitude in terms of rate constants) can be achieved between the maleimide and bromoacetyl functions when the reactions with thiols are performed at pH 6.5. The bromoacetyl function which reacts at higher pH values (e.g., pH 9.0) retained a high chemoselectivity; i.e., under conditions where it reacted appreciably with the thiols of, e.g., HS-peptides, it did react with other nucleophilic functions such as alpha- and epsilon-amino groups or imidazole, which could also be present in peptides. This differential reactivity was applied to design chemically defined and highly immunogenic liposomal diepitope constructs as synthetic vaccines, i.e., vesicles carrying at their surface two different peptides conjugated each to a specific amphiphilic anchor. This was realized by coupling sequentially at pH 6.5 and 9.0 two HS-peptides to preformed vesicles containing lipophilic anchors functionalized with maleimide and bromoacetyl groups [Boeckler, C., et al. (1999) Eur. J. Immunol. 29, 2297-2308].