Effective interactions in lysozyme aqueous solutions: a small-angle neutron scattering and computer simulation study

We report protein-protein structure factors of aqueous lysozyme solutions at different pH and ionic strengths, as determined by small-angle neutron scattering experiments. The observed upturn of the structure factor at small wavevectors, as the pH increases, marks a crossover between two different regimes, one dominated by repulsive forces, and another one where attractive interactions become prominent, with the ensuing development of enhanced density fluctuations. In order to rationalize such experimental outcome from a microscopic viewpoint, we have carried out extensive simulations of different coarse-grained models. We have first studied a model in which macromolecules are described as soft spheres interacting through an attractive r(-6) potential, plus embedded pH-dependent discrete charges; we show that the uprise undergone by the structure factor is qualitatively predicted. We have then studied a Derjaguin-Landau-Verwey-Overbeek (DLVO) model, in which only central interactions are advocated; we demonstrate that this model leads to a protein-rich/protein-poor coexistence curve that agrees quite well with the experimental counterpart; experimental correlations are instead reproduced only at low pH and ionic strengths. We have finally investigated a third, "mixed" model in which the central attractive term of the DLVO potential is imported within the distributed-charge approach; it turns out that the different balance of interactions, with a much shorter-range attractive contribution, leads in this latter case to an improved agreement with the experimental crossover. We discuss the relationship between experimental correlations, phase coexistence, and features of effective interactions, as well as possible paths toward a quantitative prediction of structural properties of real lysozyme solutions.     

Liquid-liquid extraction of lysozyme using a dye-modified ionic liquid

An affinity-dye, Cibacron Blue 3GA (CB), derivatized organic salt [BMIM]3[CB] was synthesized for lysozyme extraction. This compound was formed by mixing an ionic liquid (IL) [BMIM][Cl] and the silver salt of CB. Liquid-liquid extractions of lysozyme from the aqueous and [BMIM]3[CB] in [BMIM][PF6] solutions were examined in this study. The transfer of lysozyme from the aqueous phase to the IL phase decreased while the pH of the aqueous phase increased. An extraction higher than 90% was observed at pH 4. Under a high ionic strength, the lysozyme would transform back from the IL phase into the aqueous phase. Lysozyme molecules were almost quantitatively recovered from the IL phase to the aqueous solutions of 1M KCl under pH 9-11. It appeared that the extraction was specific for lysozyme in contrast to cytochrome c, ovalbumin, and bovine serum albumin. The extraction efficiency of the IL phase remained essentially the same after eight cycles of extraction.     

Protein interactions studied by SAXS: effect of ionic strength and protein concentration for BSA in aqueous solutions

We have studied a series of samples of bovine serum albumin (BSA) solutions with protein concentration, c, ranging from 2 to 500 mg/mL and ionic strength, I, from 0 to 2 M by small-angle X-ray scattering (SAXS). The scattering intensity distribution was compared to simulations using an oblate ellipsoid form factor with radii of 17 x 42 x 42 A, combined with either a screened Coulomb, repulsive structure factor, SSC(q), or an attractive square-well structure factor, SSW(q). At pH = 7, BSA is negatively charged. At low ionic strength, I < 0.3 M, the total interaction exhibits a decrease of the repulsive interaction when compared to the salt-free solution, as the net surface charge is screened, and the data can be fitted by assuming an ellipsoid form factor and screened Coulomb interaction. At moderate ionic strength (0.3-0.5 M), the interaction is rather weak, and a hard-sphere structure factor has been used to simulate the data with a higher volume fraction. Upon further increase of the ionic strength (I >or= 1.0 M), the overall interaction potential was dominated by an additional attractive potential, and the data could be successfully fitted by an ellipsoid form factor and a square-well potential model. The fit parameters, well depth and well width, indicate that the attractive potential caused by a high salt concentration is weak and long-ranged. Although the long-range, attractive potential dominated the protein interaction, no gelation or precipitation was observed in any of the samples. This is explained by the increase of a short-range, repulsive interaction between protein molecules by forming a hydration layer with increasing salt concentration. The competition between long-range, attractive and short-range, repulsive interactions accounted for the stability of concentrated BSA solution at high ionic strength.     

Effect of glycerol and dimethyl sulfoxide on the phase behavior of lysozyme: theory and experiments

Salt, glycerol, and dimethyl sulfoxide (DMSO) are used to modify the properties of protein solutions. We experimentally determined the effect of these additives on the phase behavior of lysozyme solutions. Upon the addition of glycerol and DMSO, the fluid-solid transition and the gas-liquid coexistence curve (binodal) shift to lower temperatures and the gap between them increases. The experimentally observed trends are consistent with our theoretical predictions based on the thermodynamic perturbation theory and the Derjaguin-Landau-Verwey-Overbeek model for the lysozyme-lysozyme pair interactions. The values of the parameters describing the interactions, namely the refractive indices, dielectric constants, Hamaker constant and cut-off length, are extracted from literature or are experimentally determined by independent experiments, including static light scattering, to determine the second virial coefficient. We observe that both, glycerol and DMSO, render the potential more repulsive, while sodium chloride reduces the repulsion.     

Relationship between interfacial forces measured by colloid-probe atomic force microscopy and protein resistance of poly(ethylene glycol)-grafted poly(L-lysine) adlayers on niobia surfaces

Adsorbed layers of "comb-type" copolymers consisting of PEG chains grafted onto a poly(l-lysine) (PLL) backbone on niobium oxide substrates were studied by colloid-probe AFM in order to characterize the interfacial forces associated with coatings of varying architectures (PEG/PLL ratios and PEG chain lengths) and their relevance to protein resistance. The steric and electrostatic forces measured varied substantially with the architecture of the PLL-g-PEG copolymers. Varying the ionic strength of the buffer solutions enabled discrimination between electrostatic and steric-entropic contributions to the net interfacial force. For high PEG grafting densities the steric component was most prominent, but at low ionic strengths and high grafting densities, a repulsive electrostatic surface force was also observed; its origin was assigned to the niobia charges beneath the copolymer, as insufficient protonated amine groups in the PLL backbone were available for compensation of the oxide surface charges. For lower grafting densities and lower ionic strengths there was a substantial attractive electrostatic contribution arising from interaction of the electrical double layer arising from the protonated amine groups, with that of the silica probe surface (as under low ionic strength conditions, the electrical double layer was thicker than the PEG layer). For these PLL-g-PEG coatings the net interfacial force can thus be a markedly varying superposition of electrostatic and steric-entropic contributions, depending on various factors. The force curves correlate with protein adsorption data, demonstrating the utility of AFM colloid-probe force measurements for quantitative analysis of surface forces and how they determine interfacial interactions with proteins. Such characterization of the net interfacial forces is essential to elucidate the multiple types of interfacial forces relevant to the interactions between PLL-g-PEG coatings and proteins and to advance interpretation of protein adsorption or repellence beyond the oversimplified steric barrier model; in particular, our data demonstrate the importance of an ionic-strength-dependent minimum PEG layer thickness to screen the electrostatic interactions of charged interfaces.     

Protein separation and enrichment by counter-current chromatography using reverse micelle solvent systems

A protein mixture consisting of myoglobin, cytochrome c, and lysozyme was separated by high-speed counter-current chromatography using a two-phase aqueous/reverse micelle-containing organic solvent system. About 50% stationary phase retention ratio was obtained in most chromatographic experiments. Separations were manipulated mainly by pH gradients that controlled the electrostatic interactions between the protein molecules and reverse micelles. Separations were further improved by incorporating an ionic strength gradient along with the pH gradient. Control of ionic strength in the aqueous solution helped fine-tune protein partitioning between the stationary and mobile phases. Although non-specific protein interactions affected baseline resolution, recovery of cytochrome c and lysozyme reached 90% and 82%. Furthermore, concentration or enrichment of these two proteins was achieved from a large-volume sample load. This technique can potentially be employed in the recovery and enrichment of proteins from large-volume aqueous solutions.     

Protein—Protein Interactions in Aqueous Ammonium Sulfate Solutions. Lysozyme and Bovine Serum Albumin (BSA)

Osmotic pressures have been measured to determine lysozyme—lysozyme,BSA—BSA, and lysosyme—BSA interactions for protein concentrations to 100 g-L^–1in an aqueous solution of ammonium sulfate at ambient temperature, as a functionof ionic strength and pH. Osmotic second virial coefficients for lysozyme, forBSA, and for a mixture of BSA and lysozyme were calculated from theosmotic-pressure data for protein concentrations to 40 g-L^–1. The osmotic second virialcoefficient of lysozyme is slightly negative and becomes more negative withrising ionic strength and pH. The osmotic second virial coefficient for BSA isslightly positive, increasing with ionic strength and pH. The osmotic second virialcross coefficient of the mixture lies between the coefficients for lysozyme andBSA, indicating that the attractive forces for a lysozyme—BSA pair areintermediate between those for the lysozyme—lysozyme and BSA—BSA pairs. For proteinconcentrations less than 100 g-L^–1, experimental osmotic-pressure data comparefavorably with results from an adhesive hard-sphere model, which has previouslybeen shown to fit osmotic compressibilities of lysozyme solutions.     

Kinetics of Lysozyme Crystallization From Solution

Abstract

We suggest that the growth of molecular aggregates is the rate-controlling step in the crystallization of lysozyme from pH buffered aqueous solutions of strong electrolytes. We propose that the aggregation reaction passes through a charged transition state whose rate of formation is accelerated by Debye-Huckel screening and whose charge is stabilized by ion exchange with the solution. Applying the theory of the “primary kinetic salt effect”, we predict that the half-life for decay of the lysozyme concentration in solution in contact with a growing crystal should decrease linearly with the square root of the ionic strength. This prediction is confirmed experimentally in the case of lysozyme crystals precipitating at 4°C from pH buffered aqueous solutions of sodium chloride.     

Mechanical properties of interfacial films formed by lysozyme self-assembly at the air-water interface

We present the first characterization of the mechanical properties of lysozyme films formed by self-assembly at the air-water interface using the Cambridge interfacial tensiometer (CIT), an apparatus capable of subjecting protein films to a much higher level of extensional strain than traditional dilatational techniques. CIT analysis, which is insensitive to surface pressure, provides a direct measure of the extensional stress-strain behavior of an interfacial film without the need to assume a mechanical model (e.g., viscoelastic), and without requiring difficult-to-test assumptions regarding low-strain material linearity. This testing method has revealed that the bulk solution pH from which assembly of an interfacial lysozyme film occurs influences the mechanical properties of the film more significantly than is suggested by the observed differences in elastic moduli or surface pressure. We have also identified a previously undescribed pH dependency in the effect of solution ionic strength on the mechanical strength of the lysozyme films formed at the air-water interface. Increasing solution ionic strength was found to increase lysozyme film strength when assembly occurred at pH 7, but it caused a decrease in film strength at pH 11, close to the pI of lysozyme. This result is discussed in terms of the significant contribution made to protein film strength by both electrostatic interactions and the hydrophobic effect. Washout experiments to remove protein from the bulk phase have shown that a small percentage of the interfacially adsorbed lysozyme molecules are reversibly adsorbed. Finally, the washout tests have probed the role played by additional adsorption to the fresh interface formed by the application of a large strain to the lysozyme film and have suggested the movement of reversibly bound lysozyme molecules from a subinterfacial layer to the interface.     

Thermosensitive behavior of poly(ethylene glycol)/poly(2‐(N,N‐dimethylamino)ethyl methacrylate) double hydrophilic block copolymers

The poly(ethylene glycol)/poly(2‐(N,N‐dimethylamino)ethyl methacrylate) (PEG/PDMAEMA) double hydrophilic block copolymers were synthesized by atom transfer radical polymerization using mPEG‐Br or Br‐PEG‐Br as macroinitiators. The narrow molecular weight distribution of PEG/PDMAEMA block copolymers was identified by gel permeation chromatography results. The thermosensitivity of PEG/PDMAEMA block copolymers in aqueous solution was revealed to depend significantly on pH, ionic strength, chain structure, and concentration of the block copolymers. By optimizing these factors, the cloud point temperature of PEG/PDMAEMA block copolymers can be limited within body temperature range (30–37 °C), which suggests that PEG/PDMAEMA block copolymers could be a good candidate for drug delivery systems. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 503–508, 2010     

The impact of ionic strength and background electrolyte on pH measurements in metal ion adsorption experiments

Our understanding of metal ion adsorption to clay minerals has progressed significantly over the past several decades, and theories have been promulgated to describe and predict the impacts of pH, ionic strength, and background solution composition on the extent of adsorption. Studies evaluating the effects of ionic strength on adsorption typically employ a broad range of background electrolyte concentrations. Measurement of pH in these systems can be inaccurate when pH values are measured with liquid junction pH probes calibrated with standard buffers due to changes in the liquid junction potential between standard, low ionic strength (0.05 M) buffers and high ionic strength solutions (>0.1 M). The objective of this research is to determine the extent of the error in pH values measured at high ionic strength, and to develop an approach for accurately measuring pH over a range of ionic strengths using a combined pH electrode. To achieve this objective, the adsorption of cobalt (10(-5) M) onto gibbsite (10 g/L) from various electrolyte solutions (0.01-1 M) was studied. The pH measurements were determined from calibrations with standard buffers and ionic strength corrected buffer calibrations. The results show a significant effect of the aqueous solution background electrolyte anion and ionic strength on pH measurement. The 0.5 and 1 M ionic strength metal ion adsorption edges shifted to lower pH with increasing ionic strength when pH was calibrated with standard buffers whereas no shift in the adsorption edges was observed when calibrated with ionic strength corrected buffers. Therefore, to obtain an accurate pH measurement, pH calibration should contain the same electrolyte and ionic strength as the samples.     

Surface forces measurements of spin-coated cellulose thin films with different crystallinity

A systematic study of the surface forces between a cellulose sphere and cellulose thin films of varying crystallinity has been conducted as a function of ionic strength and pH. Semicrystalline cellulose II surfaces and amorphous cellulose films were prepared by spin-coating of the precursor cellulose solutions onto oxidized silicon wafers before regeneration in water. Crystalline cellulose I surfaces were prepared by spin-coating wafers with aqueous suspensions of sulfate-stabilized cellulose I nanocrystals. These preparation methods produced thin, smooth films suitable for surface forces measurements. The interaction with the cellulose I was monotonically repulsive at pH 3.5, 5.8, and 8.5 and at 0.1, 1, and 10 mM ionic strengths. This was attributed to the presence of strongly ionizable sulfur-containing groups on the cellulose nanocrystal surfaces. The amorphous film typically showed a steric interaction up to 100 nm away from the interface that was independent of the solution conditions. A range of surface forces were successfully measured on the semicrystalline cellulose II films; attractive and repulsive regimes were observed, depending on pH and ionic strength, and were interpreted in terms of van der Waals and electrostatic interactions. Clearly, the forces acting near cellulose surfaces are very dependent on the way the cellulose surface has been prepared.     

Analysis of pH-dependent protein interactions with gel filtration medium.

A prepacked Superose 12 HR 10/30 column was used to study the effects of elution ionic strength and pH on the chromatographic behaviour of a strong hydrophobic Clostridium thermocellum endoglucanase (1) and two weak hydrophobic proteins, Clostridium thermocellum endoglucanase C and egg white lysozyme. Ion-exclusion or ion-exchange interactions between weakly hydrophobic proteins and the gel matrix were observed at low ionic strength, depending on whether the pH of the elution buffer was higher or lower than the pI values of the proteins. These interactions were due to the presence of negatively charged groups on the surface of Superose and could be eliminated at any pH by adding electrolyte at a concentration determined by its chemical identity. The optimum results were observed with sodium sulphate at a concentration of 100 mM. The chromatographic behaviour of strong hydrophobic endoglucanase (1) on a Superose column as a function of pH was much more complex because of two interplaying effects, electrostatic and hydrophobic. Ideal size-exclusion chromatography could be achieved only in a narrow range of the conditions: first, the mobile phase must contain a weak salting-out electrolyte such as NaCl, and second, the mobile phase pH must be high enough that hydrophobic interactions between the solute and support are balanced by their electrostatic repulsion. At pH greater than pI, the retardation of endoglucanase (1) gradually increased with decreasing pH as a result of lowering of repulsive electrostatic interactions whether or not the buffer ionic strength was high. At pH less than pI a drastic increase in the capacity factor k' was observed owing to the additivity of hydrophobic and ion-exchange effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adhesion of polyether-modified poly(acrylic acid) to mucin

Pluronic-PAA, a thermogelling copolymer composed of side chains of poly(acrylic acid) (PAA) grafted onto a backbone of Pluronic copolymer, is of interest as a vehicle for the controlled release of compounds. An important feature of such a vehicle is its bioadhesive/mucoadhesive properties, which in the case of Pluronic-PAA are significant due to the presence of the PAA side chains. An atomic force microscopy (AFM) method has been developed and utilized to investigate the interactions between a Pluronic-PAA-modified microsphere and mucous substrates. The bioadhesive force was successfully measured, and trends were observed under conditions of varying pH and ionic strength. Pluronic-PAA exhibits significant mucoadhesion over a range of pH values, with mucoadhesion being optimal at pH 4-5 (adhesive force approximately 80 mN/cm(2)) and dropping sharply at higher pH, to a value of approximately 20 mN/cm(2) at pH 8. The mucoadhesive force decreased with increasing ionic strength, from a value of approximately 80 mN/cm(2) in 0.025 M NaCl to approximately 25 mN/cm(2) in 1.0 M NaCl. These results have been interpreted in terms of the effect of changing pH and ionic strength on electrostatic interactions and swelling of the polymer and mucin layers. Tensiometric force measurements indicated that hydrophobic interactions, as well as hydrogen bonding and electrostatic interactions, were significant in the mucoadhesion of Pluronic-PAA copolymers. Experiments with a range of Pluronic-PAA copolymers with varying PPO contents in the Pluronic segments showed that increasing the overall PPO content increased the hydrophobicity of the polymer solutions. This was reflected in the increases in the advancing contact angles with the mucin layer, indicating that hydrophobic interactions play a role in the adhesion of Pluronic-PAA to mucin.     

Polyelectrolyte–protein interaction at low ionic strength: required chain flexibility depending on protein average charge

The effect of low ionic strength leading to reduced polyelectrolyte–protein interactions has been shown by in silico and in vitro experiments, suggesting polyelectrolyte rigidity increasing at low ionic strength, thus leading to reduced interactions with proteins. This contribution elucidates polyelectrolyte–protein precipitation in the 0–2.6-mS?cm^?1 ionic strength regime with polyelectrolyte rigidity determinations, using viscosimetry at these conditions, also considering protein charge distributions, using different proteins. Precipitation yields increased from 5 to 40 % at low ionic strength to up to 90 % at intermediate ionic strength, depending on protein and polyelectrolyte type, using lysozyme and three different monoclonal antibodies. Comparing precipitation behavior of the monoclonal antibodies, a qualitative correlation between required polyelectrolyte flexibility to enhance protein precipitation and protein average charge as well as hydrophobicity of the antibodies was discovered. Antibodies with lower average charge and less hydrophobicity required more flexible polyelectrolytes to enhance precipitation behavior by allowing interaction of the polyelectrolytes with proteins, attaching to positively charged protein patches while “circumnavigating” negatively charged protein areas. In contrast, antibodies with higher protein average charge showed increasing precipitation yields up to 90 % already at lower ionic strength, associated with then more rigid polyelectrolyte structures. Therefore, designing polyelectrolytes with specific chain flexibility could help to improve precipitation behavior toward specific target proteins in polyelectrolyte-driven purification techniques.     

Effect of surface chemistry, solution pH, and ionic strength on the removal of herbicides diuron and amitrole from water by an activated carbon fiber

A study was conducted on the effects of carbon surface chemistry, solution pH, and ionic strength on the removal of diuron and amitrole from aqueous solutions by adsorption on an as-received and oxidized activated carbon fiber. Results obtained were explained by the surface characteristics of the adsorbents and the characteristics of the herbicide molecules. Under the experimental conditions used, diuron uptake was much higher than that of amitrole, despite its larger molecular dimensions, due to the lesser water solubility, greater hydrophobicity, and larger dipolar moment of diuron compared with amitrole. Uptake variations associated with differences in carbon surface oxidation, solution pH, and ionic strength were explained by corresponding changes in electrostatic, hydrophobic, and van der Waals interactions.     

Theoretical consideration of osmotic pressure in aqueous protein/salt systems based on extended hard core Lennard-Jones potential

A simple and analytical pair potential function was developed to represent the osmotic pressures in aqueous protein/salt systems under various conditions. Based on a hard core Lennard-Jones (HCLJ) potential model, the new potential function considers various interactions by extending the attractive Lennard-Jones potential. A temperature-dependent coefficient term was introduced to take into account the specific properties of given materials. Comparison of the new potential function with the HCLJ model in hydrocarbon and water systems showed that consideration of the temperature dependence in the potential function was effective, especially for strong polar systems such as water. To predict the osmotic pressures of aqueous lysozyme/(NH(4))(2)SO(4) solutions of various ionic strength and pH, the energy parameters of lysozyme were correlated with the experimental cloud point temperature. The proposed model agreed fairly well with the experimental osmotic pressure data with only previously obtained parameters.     

Application of sucrose fatty acid ester to reverse micellar extraction of lysozyme

Reverse micellar extraction of lysozyme has been carried out using an organic solution containing a mixture of monoester and polyester of sucrose fatty acid ester. The forward extraction of lysozyme from the feed aqueous phase to the reverse micellar organic phase of the mixture of monoester and polyester of sucrose fatty acid ester at pH 7.2 was strongly dependent upon the weight fraction of monoester, while any amount of lysozyme was not extracted only by using monoester or polyester. The forward extraction ratio dramatically increased with an increase in the concentration of fatty acid ester, and was high around neutral pH and at low ionic strength. The backward extraction of lysozyme from the reverse micellar organic phase to the recovery aqueous phase exhibited high efficiency at acidic pH value or at high ionic strength. The addition of sucrose into the recovery aqueous phase promoted the backward extraction ratio, and caused the activity of lysozyme recovered from the reverse micellar phase to be retained perfectly.     

Effect of anionic structure on the phase formation and hydrophobicity of amino acid ionic liquids aqueous two-phase systems

Compared with the conventional ionic liquids, amino acid ionic liquids are more biodegradable and biocompatible, and can enhance stability of biomaterials. In this work, amino acid ionic liquids 1-butyl-3-methylimidazolium L-serine ([C(4)mim][Ser]), 1-butyl-3-methylimidazolium glycine ([C(4)mim][Gly]), 1-butyl-3-methylimidazolium L-alanine ([C(4)mim][Ala]) and 1-butyl-3-methylimidazolium L-leucine ([C(4)mim][Leu]) have been synthesized. These ionic liquids are found to form aqueous two-phase systems (ATPSs) by the salted-out of K(3)PO(4) in aqueous solutions. Phase diagram of the ATPSs and the Gibbs energies of transfer of methylene group from the bottom salt-rich phase to the top ionic liquid-rich phase have been determined at 298.15K and pH 14, and the effect of anionic structure of the ionic liquids on phase formation of the ATPSs and the relative hydrophobicity between the top and the bottom phases are then examined. In order to understand the effect of relative hydrophobicity of the phases in equilibrium in the ATPSs on the extraction/separation capability of biomolecules, the partition coefficients of cytochrome-c (as a model biomolecule) in the ATPSs are measured by spectrophotometry. It is suggested that hydrophobic interactions are mainly responsible for the higher partition coefficients of cytochrome-c in aqueous two-phase systems at pH 14, and the extraction and separation capacity of biomolecules can be improved by the modulation of the relative hydrophobicity of the phases and/or the pH of the system.     

Electrolyte and pH effects on Pb(II)-calcite sorption processes: the role of the PbCO0 (3aq) complex

Batch sorption experiments were conducted under conditions of ambient temperature and atmospheric PCO2g to determine the effects of electrolyte type, ionic strength, and pH on Pb(II) interactions with calcite. For 0.15 M nitrate and chloride solutions at pH 8.2, no significant effect of electrolyte type on Pb sorption was observed. Varying ionic strength from 0.15 to 0.5 M produced little effect on Pb sorption in nitrate compared to chloride solutions in which Pb uptake decreased with increasing ionic strength. For a pH range of 7.3-9.4 in 0.15-0.2 M nitrate solutions, Pb sorption increased from pH 7.3 to 8.5 with a subsequent decrease in uptake out to pH 9.4. The trends in electrolyte and pH experiments correlate well with those for PbCO0(3aq) speciation, indicating that this metal-ligand complex in solution dictates Pb sorption in the system under investigation.