Possible origin of the inverse and direct Hofmeister series for lysozyme at low and high salt concentrations

Protein solubility studies below the isoelectric point exhibit a direct Hofmeister series at high salt concentrations and an inverse Hofmeister series at low salt concentrations. The efficiencies of different anions measured by salt concentrations needed to effect precipitation at fixed cations are the usual Hofmeister series (Cl(-) > NO(3)(-) > Br(-) > ClO(4)(-) > I(-) > SCN(-)). The sequence is reversed at low concentrations. This has been known for over a century. Reversal of the Hofmeister series is not peculiar to proteins. Its origin poses a key test for any theoretical model. Such specific ion effects in the cloud points of lysozyme suspensions have recently been revisited. Here, a model for lysozymes is considered that takes into account forces acting on ions that are missing from classical theory. It is shown that both direct and reverse Hofmeister effects can be predicted quantitatively. The attractive/repulsive force between two protein molecules was calculated. To do this, a modification of Poisson-Boltzmann theory is used that accounts for the effects of ion polarizabilities and ion sizes obtained from ab initio calculations. At low salt concentrations, the adsorption of the more polarizable anions is enhanced by ion-surface dispersion interactions. The increased adsorption screens the protein surface charge, thus reducing the surface forces to give an inverse Hofmeister series. At high concentrations, enhanced adsorption of the more polarizable counterions (anions) leads to an effective reversal in surface charge. Consequently, an increase in co-ion (cations) adsorption occurs, resulting in an increase in surface forces. It will be demonstrated that among the different contributions determining the predicted specific ion effect the entropic term due to anions is the main responsible for the Hofmeister sequence at low salt concentrations. Conversely, the entropic term due to cations determines the Hofmeister sequence at high salt concentrations. This behavior is a remarkable example of the charge-reversal phenomenon.     

Liquid-liquid coexistence surface for lysozyme: role of salt type and salt concentration

The liquid-liquid phase separation curves for lysozyme in a salt solution are known to depend on salt type and salt concentration. For the case of monovalent cations, the cloud point temperature typically increases with increasing salt concentration, for fixed lysozyme concentration. For the case of divalent cations, however, a maximum in the cloud point temperature is observed that has been interpreted as being due to ion binding to the protein surface and subsequent water structuring. In this paper, we use a simple square well model due to Grigsby et al. (Biophys. Chem. 2001, 91, 231-243), whose well depth depends on salt type and salt concentration, to determine the phase coexistence surfaces from experimental data. The surfaces are shown as a function of temperature, salt concentration, and protein concentration for two typical salts, NaCl and MgCl2. These surfaces are calculated using the results of a single standard Monte Carlo simulation and a simple scaling argument and are in reasonably good agreement with known experimental results.     

Net charge and electrophoretic mobility of lysozyme charge ladders in solutions of nonionic surfactant

We report on the electrophoretic mobility and on the thermal diffusion of lysozyme proteins dissolved in aqueous solutions of a nonionic surfactant (C12E6) at a wide range of concentrations of the surfactant (0-20% by weight). We want to estimate the influence of a dense network of elongated micelles of C12E6 on the effective charge of the proteins as observed in the capillary electrophoresis experiments. The possible mechanism leading to the change in the effective charge of protein could involve the deformation of the cloud of counterions around the protein when it squeezes through the narrow (of the order of a protein diameter) aqueous channels formed in the solution of elongated micelles. The combination of independent measurements of the electrophoretic mobility of a family of modified proteins (lysozyme charge ladder [Colton et al. J. Am. Chem. Soc. 1997, 119, 12701]), of the microviscosity of the solutions of surfactant (obtained via fluorescence correlation spectroscopy), and of the hydrodynamic radius of the proteins (photon correlation spectroscopy) allow us to conclude that the effective charge of the proteins is not affected by the presence of surfactant, even at high concentrations.     

Regulation of electroosmotic flow and electrophoretic mobility of proteins for concentration without desalting

Proteins were concentrated and separated in 0.6% poly(ethylene oxide) (PEO) solution using a capillary filled with Tris-borate (TB) buffer prior to analysis and detected by laser-induced native fluorescence using a pulsed Nd:YAG laser. During the concentration and separation, PEO solution entered the capillary by electroosmotic flow. When proteins dissolved in high salts (phosphate-buffered saline) were separated using 0.6% PEO solution prepared in 200 mM TB buffer, pH 9.0, the limits of detection (LODs) at signal-to noise ratios=3 for carbonic anhydrase (CA) and alpha-lactalbumin (alpha-lac) were on the levels of sub microM and microM, respectively. The LOD values compared to those obtained in 38 mM TB buffer were relatively high, which is likely due to salt quenching, Joule heating and poor stacking. To improve sensitivity for analysis of proteins in high-conductivity media, two on-line concentration approaches without desalting were developed. When using a capillary filled with 1.5 M TB buffer, pH 10.0, and PEO solution prepared in 800 mM TB buffer, pH 9.0, the LOD values for CA and alpha-lac were 13.8 nM and 126.0 nM, respectively, which were about 4.7 and 11.2-fold sensitivity enhancements compared to those obtained by a conventional hydrodynamic injection (30 cm height for 10 s), respectively. The sensitivity was further improved by injecting a short plug of low pH buffer after protein injection using a capillary filled with 1.5 M TB buffer, pH 10.0, and PEO solution prepared in 400 mM TB buffer, pH 9.0. A linear relationship between the peak height and the injection volume up to 0.81 microl was obtained and the LOD values for CA and alpha-lac were down to 4.7 and 37.8 nM.     

Distinct conformations of DNA-stabilized fluorescent silver nanoclusters revealed by electrophoretic mobility and diffusivity measurements

Silver-DNA nanoclusters (Ag:DNAs) are novel fluorophores under active research and development as alternative biomolecular markers. Comprised of a few-atom Ag cluster that is stabilized in water by binding to a strand of DNA, they are also interesting for fundamental explorations into the properties of metal molecules. Here, we use in situ calibrated electrokinetic microfluidics and fluorescence correlation spectroscopy to determine the size, charge, and conformation of a select set of Ag:DNAs. Among them is a pair of spectrally distinct Ag:DNAs stabilized by the same DNA sequence, for which it is known that the silver cluster differs by two atoms. We find these two Ag:DNAs differ in size by ～30%, even though their molecular weights differ by less than 3%. Thus a single DNA sequence can adopt very different conformations when binding slightly different Ag clusters. By comparing spectrally identical Ag:DNAs that differ in sequence, we show that the more compact conformation is insensitive to the native DNA secondary structure. These results demonstrate electrokinetic microfluidics as a practical tool for characterizing Ag:DNA.     

Characterization of globular protein solutions by dynamic light scattering, electrophoretic mobility, and viscosity measurements

In this work, physicochemical properties of two globular proteinsbovine serum albumin (BSA) having a molecular weight of 67 kDa and human serum albumin (HSA) having a molecular weight of 69 kDawere characterized. The bulk characteristics of these proteins involved the diffusion coefficient (hydrodynamic radius), electrophoretic mobility, and dynamic viscosity as a function of protein solution concentration for various pH values. The hydrodynamic radius data suggested an association of protein molecules, most probably forming compact dimers. Using the hydrodynamic diameter and the electropheretic mobility data allowed the determination of the number of uncompensated (electrokinetic) charges on protein surfaces. The electrophoretic mobility data were converted to zeta potential values, which allowed one to determine the isoelectric point (iep) of these proteins. It was found to be at pH 5.1 for both proteins, in accordance with previous experimental data and theoretical estimations derived from amino acid composition and p K values. To determine further the stability of protein solutions, dynamic viscosity measurements were carried out as a function of their bulk volume concentration for various pH values. The intrinsic viscosity derived from these measurements was interpreted in terms of the Brenner model, which is applicable to hard spheroidal particles. It was found that the experimental values of the intrinsic viscosity of these proteins were in good agreement with this model when assuming protein dimensions of 9.5 x 5 x 5 nm3 (prolate spheroid). The possibility of forming linear aggregates of association degree higher than 2 was excluded by these measurements. It was concluded that the combination of dynamic viscosity and dynamic light scattering can be exploited as a convenient tool for detecting not only the onset of protein aggregation in suspensions but also the form and composition of these aggregates.     

On the adsorption of IgG onto polystyrene particles: electrophoretic mobility and critical coagulation concentration

An experimental investigation on the adsorption of immuno -globulin molecules on polystyrene microspheres is described. Three different IgG samples were adsorbed on latex particles. One was of polyclonal nature with a broad range of isoelectric points (6.1–8.7), whereas the other samples were of monoclonal nature, Mab 1 and Mab 2 with i.e.p. of (5.65±0.15) and (7.7±0.1), respectively. Adsorption isotherms at different ionic strengths and pH were performed. Most of the adsorption isotherms showed well-defined plateaus. Because of instability in solution of Mab 2 in the pH values of 7 and 8, no plateau values were found in the adsorption isotherms at both pH-values. Maximum protein adsorption was found around the i.e.p. of the protein. According to the findings, the IgG adsorption on polystyrene surface is strongly irreversible with respect to pH changes. The ionic-strength changes, however, exert a pronounced effect on the adsorption-desorption processes of IgG on negatively charged polystyrene surface. Also, electrophoresis experiments were performed to gain information on the electrostatic interaction between the IgG molecules and the PS latex. With increasing the adsorbed amount of IgG the absolute value of mobility decreases to reach a plateau value. The isoelectric pH of the IgG-PS complex is always smaller than the i.e.p. of the dissolved IgG, indicating that the PS surface charge must partly compensate the positive charge on the protein. Finally, the colloidal stability of the rabbit IgG/PS complex is always very low, whereas the Mab/PS complexes are very stable when the charge electrokinetically mobilized by these systems is very large.     

Effects of temperature and salt concentration on the structural stability of human lymphotactin: insights from molecular simulations

Extensive molecular dynamics (MD) simulations ( approximately 70 ns total) with explicit solvent molecules and salt ions are carried out to probe the effects of temperature and salt concentration on the structural stability of the human Lymphotactin (hLtn). The distribution of ions near the protein surface and the stability of various structural motifs are observed to exhibit interesting dependence on the local sequence and structure. Whereas chloride association to the protein is overall enhanced as the temperature increases, the sodium distribution in the C-terminal helical region and, to a smaller degree, the chloride distribution in the same region are found higher at the lower temperature. The similar trend is also observed in nonlinear Poisson-Boltzmann calculations with a temperature-dependent water dielectric constant, once conformational averaging over a series of MD snapshots is done. The unexpected temperature dependence in the ion distribution is explained on the basis of the cancellation of association entropy for ion-side chain pairs of opposite-charge and like-charge characters, which have positive and negative contributions, respectively. The C-terminal helix is observed to partially melt whereas a short beta strand forms at the higher temperature with little salt dependence. The N-terminal region, by contrast, develops partial helical structure at a higher salt concentration. These observed behaviors are consistent with solvent and salt screening playing an important role in stabilizing the canonical chemokine fold of hLtn.     

Colloidal stability of aqueous polymeric dispersions: effect of pH and salt concentration

Aqueous film coatings often contain some electrolytes, organic acids, and pigments to give functions of sustained release, time-controlled release, or protection against light. Additions of some electrolytes or organic acids into latex dispersion for an aqueous film coating affect its colloidal stability. We characterized the aqueous polymeric latexes used in the pharmaceutical industry by measuring zeta potential and particle size, and evaluated this colloidal stability using DLVO theory. Three polymethacrylate-based aqueous polymeric latexes, Eudragit L30D-55, Eudragit RS30D and Eudragit NE30D, having anionic, cationic, and neutral polymer, respectively, were used in this study. The Hamaker constant of the polymethacrylate-based latex was determined to be 6.35 x 10(-21) J, and the total potential energy of the latex dispersion was calculated. The total potential energy of interaction between pairs of latex particles changes by altering the salt concentration and pH. The experimental results of stability in the anionic and the cationic latex dispersions can be explained by the total interaction energies. However, the stabilization of the neutral latex did not match the calculated result. The steric interaction produced by the surfactant likely resulted in the stable dispersion of this latex.     

Coupling between lysozyme and glycerol dynamics: microscopic insights from molecular-dynamics simulations

We explore possible molecular mechanisms behind the coupling of protein and solvent dynamics using atomistic molecular-dynamics simulations. For this purpose, we analyze the model protein lysozyme in glycerol, a well-known protein-preserving agent. We find that the dynamics of the hydrogen bond network between the solvent molecules in the first shell and the surface residues of the protein controls the structural relaxation (dynamics) of the whole protein. Specifically, we find a power-law relationship between the relaxation time of the aforementioned hydrogen bond network and the structural relaxation time of the protein obtained from the incoherent intermediate scattering function. We demonstrate that the relationship between the dynamics of the hydrogen bonds and the dynamics of the protein appears also in the dynamic transition temperature of the protein. A study of the dynamics of glycerol as a function of the distance from the surface of the protein indicates that the viscosity seen by the protein is not the one of the bulk solvent. The presence of the protein suppresses the dynamics of the surrounding solvent. This implies that the protein sees an effective viscosity higher than the one of the bulk solvent. We also found significant differences in the dynamics of surface and core residues of the protein. The former is found to follow the dynamics of the solvent more closely than the latter. These results allowed us to propose a molecular mechanism for the coupling of the solvent-protein dynamics.     

Coupling between lysozyme and trehalose dynamics: microscopic insights from molecular-dynamics simulations

We have carried out molecular-dynamics simulations on fully flexible all-atom models of the protein lysozyme immersed in trehalose, an effective biopreservative, with the purpose of exploring the nature and extent of the dynamical coupling between them. Our study shows a strong coupling over a wide range of temperatures. We found that the onset of anharmonic behavior was dictated by changes in the dynamics and relaxation processes in the trehalose glass. The physical origin of protein-trehalose coupling was traced to the hydrogen bonds formed at the interface between the protein and the solvent. Moreover, protein-solvent hydrogen bonding was found to control the structural relaxation of the protein. The dynamics of the protein was found to be heterogeneous; the motions of surface and core atoms had different dependencies on temperature and, in addition, the surface atoms were more sensitive to the dynamics of the solvent than the core atoms. From the solvent perspective we found that the dynamics near the protein surface showed an unexpected enhanced mobility compared to the bulk. These results shed some light on the microscopic origins of the dynamical coupling in protein-solvent systems.     

Effects of pH and salt concentration on oil-in-water emulsions stabilized solely by nanocomposite microgel particles

Aqueous dispersions of lightly cross-linked poly(4-vinylpyridine)/silica nanocomposite microgel particles are used as a sole emulsifier of methyl myristate and water (1:1 by volume) at various pH values and salt concentrations at 20 degrees C. These particles become swollen at low pH with the hydrodynamic diameter increasing from 250 nm at pH 8.8 to 630 nm at pH 2.7. For batch emulsions prepared at pH 3.4, oil-in-water (o/w) emulsions are formed that are stable to coalescence but exhibit creaming. Below pH 3.3, however, these emulsions are very unstable to coalescence and rapid phase separation occurs just after homogenization (pH-dependent). The pH for 50% ionization of the pyridine groups in the particles in the bulk (pK(a)) was determined to be 3.4 by acid titration measurements of the aqueous dispersion. Thus, the charged swollen particles no longer adsorb at the oil-water interface. For continuous emulsions (prepared at high pH with the pH then decreased abruptly or progressively), demulsification takes place rapidly below pH 3.3, implying that particles adsorbed at the oil-water interface can become charged (protonated) and detached from the interface in situ (pH-responsive). Furthermore, at a fixed pH of 4.0, addition of sodium chloride to the aqueous dispersion increases the degree of ionization of the particles and batch emulsions are significantly unstable to coalescence at a salt concentration of 0.24 mol kg(-1). The degree of ionization of such microgel particles is a critical factor in controlling the coalescence stability of o/w emulsions stabilized by them.     

Refolding human lysozyme produced as an inclusion body by urea concentration and pH gradient ion exchange chromatography

Summary A new dual-gradient ion exchange chromatographic method was developed to improve the refolding yield of human lysozyme produced inEscherichia coli as an inclusion body. The dissolved and stretched polypeptide chain in a concentrated non-ionic denaturant was adsorbed onto an ion exchange column and induced to refold by gradually decreasing the denaturant concentration and increasing pH in the flowing buffer. The dual gradients of denaturant concentration and pH provided a gradual change of the solution environment along the chromatographic column for the protein to refold, resulting in enhanced activity yield and purity. A post-separation was also studied using size-exclusion chromatography to remove protein aggregates and mis-folded proteins after the refolding step.     

Unprecedented tunable hydrophobic effect and anion recognition triggered by AIE with Hofmeister series in water

An unprecedented tunable hydrophobic effect in self-assembly of a small cationic organic fluorophore(NI-TPy~+)-based with aggregation-induced emission(AIE) property was realized in aqueous solution.The amplification of hydrophobicity was found to be significantly dependent upon the increasing aggregate s of NI-TPy~+,which enable d the study of the hydrophobic binding of chaotropic anions with the Hofmeister series.     

Photophysics of a series of efficient fluorescent pH probes for dual-emission-wavelength measurements in aqueous solutions

This paper evaluates the 5-aryl-2-pyridyloxazole backbone to engineer donor-acceptor fluorescent pH probes after one- or two-photon absorption. Parent fluorophores, as well as derivatives that can be used to label biomolecules, can be easily obtained in good yields. These molecules exhibit a large one-photon absorption in the near-UV range, and a strong fluorescence emission that covers the whole visible domain. The 5-aryl-2-pyridyloxazole derivatives also possess significant cross sections for two-photon absorption. Upon pyridine protonation, large shifts were observed in the absorption spectra after one- and two-photon excitation, as well as in the emission spectra. This feature was used to measure the pK(a) of the investigated compounds that range between 2 and 8. In most of the investigated derivatives, the pK(a) increased upon light excitation and protonation exchanges took place during the lifetime of the excited state, as shown by phase-modulation fluorometry analysis. Several 5-aryl-2-pyridyloxazole derivatives are suggested as efficient probes to reliably measure the pH of aqueous solutions by means of ratiometric methods that are dependent on fluorescence emission.     

Simultaneous measurements of the electrophoretic mobility,diffusion coefficient and orientation of dsDNA during electrophoresis in polymer solutions

We determined simultaneously the electrophoretic mobility, diffusion coefficient D and molecular orientation during electrophoresis of dsDNAs in polymer solutions ranging from the dilute to the semidilute regime. We established, for the first time, master scaling laws for the diffusion coefficient showing a universal behavior. A model found in the literature designed for the dilute regime allows, surprisingly, to describe the mobility data over the whole range of concentrations studied and at the same time the biased reptation with fluctuations (BRF) failed for the semidilute regime, even when constraint release of the network was taken into account. These quantitative determinations of D are of practical interest to evaluate band broadening during capillary electrophoresis and provide data for stimulating investigation of the physics of DNA electrophoretic motion.     

Analysis of the interfacial properties of fibrillated and nonfibrillated oral streptococcal strains from electrophoretic mobility and titration measurements: evidence for the shortcomings of the 'classical soft-particle approach'

Chemical and structural intricacies of bacterial cells complicate the quantitative evaluation of the physicochemical properties pertaining to the cell surface. The presence of various types of cell surface appendages has a large impact on those properties and therefore on various interfacial phenomena, such as aggregation and adhesion. In this paper, an advanced analysis of the electrophoretic mobilities of fibrillated and nonfibrillated strains (Streptococcus salivarius HB and Streptococcus salivarius HB-C12, respectively) is performed over a wide range of pH and ionic strength conditions on the basis of a recent electrokinetic theory for soft particles. The latter extends the approximate formalism originally developed by Ohshima by solving rigorously the fundamental electrokinetic equations without restrictions on the bacterial size, charge, and double layer thickness. It further allows (i) a straightforward implementation of the dissociation characteristics, as evaluated from titration experiments, of the ionogenic charged groups distributed throughout the bacterial cell wall and/or the surrounding exopolymer layer and (ii) the inclusion of possible specific interactions between the charged groups and ions from the background electrolyte other than charge-determining ions. The theory also enables an estimation of possible swelling/shrinking processes operating on the outer polymeric layer of the bacterium. Application of the electrokinetic model to HB and HB-C12 clearly shows a significant discrepancy between the amount of surface charges probed by electrophoresis and by protolytic titration. This is ascribed to the specific adsorption of cations onto pristine charged sites in the cell wall. Physicochemical parameters pertaining to the hydrodynamics (softness degree) and electrostatics of the bacterial cell wall (HB-C12) and soft polymeric layer (HB) are quantitatively derived.     

Phase equilibria for salt-induced lysozyme precipitation: Effect of salt type and temperature

The salt-induced precipitation of lysozyme from aqueous solutions was studied through precipitation assays in which the equilibrium compositions of the coexisting phases were determined. Lysozyme precipitation experiments were carried out at 5, 15 and 25 °C and pH 7.0 with ammonium sulfate, sodium sulfate and sodium chloride as precipitating agents. In these experiments a complete separation of the coexisting phases (liquid and solid) could not be achieved. Nevertheless it was possible to determine the composition of the precipitate. The enzymatic activity of lysozyme in the supernatant phase as well as in the precipitate phase was also determined. The activity balance suggests that there is a relationship between the composition of the true precipitate and the total activity recovery.     

Effects of the limited analyte solubility on its mobility and zone shape: electrophoretic behavior of sanguinarine and chelerythrine around pH 7

Electrophoretic mobilities and shapes of zones of sanguinarine and chelerythrine in aqueous media around pH 7 are affected by limited solubility of their uncharged forms and by the pH-dependent chemical equilibrium between cationic and uncharged forms of these alkaloids. The sanguinarine solubility in sodium MOPS of pH 7.4 was estimated at 50 micromol x L(-1). Sanguinarine zones in this buffer have the shape of tailed peak with concentration-independent mobility if the injected sanguinarine concentration exceeds this solubility limit only slightly. The chelerythrine solubility is higher because of lower dissociation constants of its cations. Precipitation of sanguinarine and chelerythrine with the phosphate anions decelerates their electrophoretic transport in phosphate buffer. Sanguinarine solubility is 5 micromol x L(-1) at the most in 13 mmol x L(-1) sodium phosphate buffer of pH 7.4. Acidifying of the sample up to pH 3 decreases the tailing of the peaks of sanguinarine and chelerythrine and contributes to the rise of sharp maxima of their migrating zones. Any capillary coating deteriorates the peak shape.     

Effective interaction between two ion-adsorbing plates: Hofmeister series and salting-in/salting-out phase diagrams from a global mean-field analysis

Interactions between ions and solutes are key to ion-specificity. A generic model in which ions interact via square well potentials of finite range with charged plates is solved analytically on the Poisson-Boltzmann level and analyzed globally for varying surface charge, salt concentration, and ion-surface affinity. Ion adsorption as well as depletion can lead to stably bound plates at finite separation, relevant for the equilibrium salting-out of small solutes such as proteins. The interplate pressure at large plate separation, relevant for aggregation kinetics of large solutes, exhibits direct as well as indirect Hofmeister ordering, depending on surface charge and salt concentration. A simple method for mapping explicit ion-surface potentials of mean force as obtained from solvent-explicit molecular dynamics simulations onto square-well potential parameters is demonstrated.