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.     

Hofmeister effects on poly(NIPAM) microgel particles: macroscopic evidence of ion adsorption and changes in water structure.

The term Hofmeister effects is broadly used to refer to ionic specificities in many different physical, chemical and biological phenomena. The origin of this ionic specificity is sought in two interdependent microscopic sources: 1) the peculiarities of the solvent structure near surfaces and around the ions, and 2) specific ion adsorption-exclusion mechanisms near a surface. In this work, Hofmeister effects on poly(N-isopropylacrylamide) [poly(NIPAM)]-based microgels are examined. Poly(NIPAM) particles are thermally sensitive microgels exhibiting volume-phase transitions with temperature. This temperature-sensitive system seems to be suitable for the independent observation of the two microscopic sources of Hofmeister effects. On the one hand, volume-phase transition, evaluated by photon correlation spectroscopy (PCS), gives information about how the presence of ions changes the water structure around the poly(NIPAM) chains. On the other hand, electrokinetic studies show relevant data about ionic adsorption-exclusion phenomena at the polymer surface.     

Hofmeister effects at low salt concentration due to surface charge transfer

We present a theoretical comparison of the surface forces between two graphite-like surfaces at salt concentrations below 10 mM with surfaces charged by various mechanisms. Surface forces include a surface charging or chemisorption contribution to the total free energy. Surfaces are charged by charge regulation (H⁺ binding), site competition (H⁺ and cation binding) and redox charging with electrodes coupled to a countercell. Constant surface charge is also considered. Surface parameters are calibrated to give the same potential when isolated. Nonelectrostatic physisorption energies of the potential determining ions provide a specific and significant contribution to the charging energy. Consequently ion specificity is found in the surface forces at concentrations of 1–10 mM, which is not observed under constant charge conditions. The force between redox electrodes continues to show Hofmeister effects at 0.01 mM. We refer to this low concentration Hofmeister effect as “Hofmeister charging”, and suggest that the more common high concentration ion specific effects may be known as “Hofmeister screening”. Hofmeister series are considered over LiCl, NaCl, KCl and NaNO₃, NaClO₄, NaSCN with the cations (or H⁺) being the potential determining ions. A K⁺ anomaly is attributed to the small size of the weakly hydrated chaotropic K⁺ ion, with Li⁺ and Na⁺ explicitly modelled as strongly hydrated cosmotropes.     

Hofmeister离子对水溶液中热响应聚合物的局域环境的影响

The Hofmeister ion effect is a very interesting but elusive phenomenon, the importance of which is revealed in self-assembly, ion recognition, and protein folding regulation. With an increasing number of studies suggesting that interactions between ions and solutes play a role in the Hofmeister ion effect, the nature of the Hofmeister phenomenon becomes more debatable. Yet, it is not clear whether the Hofmeister ion effect is a local effect or bulk effect that can reach beyond many hydration shells, where specific interactions between ions and solutes play key roles. In order to further explore this, we applied proton nuclear magnetic resonance (¹H-NMR) spectroscopy to study the effects of specific ions on the local environment around N, N-dimethylpropionamide (NDA) and N-isopropylisobutyramide (NPA), which are the model compounds for poly(2-ethyl-2-oxazoline) and poly(N-isopropylacrylamide), respectively. These polymers are important bio-engineering materials that possess thermoresponsive properties and are also subject to specific ion effects. By correlating the changes in chemical shifts of the two methyl groups on either side of the amide bond, it was found that the Hofmeister ion effects on NPA were more anisotropic than on NDA, and that the cationic effects were more anisotropic than the anionic effects on NPA. These results indicated that the effects of specific ions were almost identical for all methyl groups of NDA. On the other hand, NPA is a larger molecule; thus, not all of its methyl groups were subjected to the specific ion effects to the same extent. The calculation of the electrostatic potential surfaces of NDA and NPA suggested that these observations on the Hofmeister ion effects might be due to steric hindrance, and that the observations on the cationic effects might be due to the interactions between cations and NPA being stronger than the interactions between anions and NPA. This would explain why the highly charged cations caused a significant anisotropicity. Additionally, we found that the chemical shift of the water protons (Δδ_H2O) of conventional kosmotropic anions was larger than zero, which suggested a stronger HB and more charge transfer between water and these anions. The Δδ_H2O of conventional chaotropic anions was less than zero. Despite the different solutes, the results were indifferent in both NDA and NPA solutions. Surprisingly, the Δδ_H2O of Cl^- at concentrations lower than 1 mol∙L^-1 was zero, thus becoming the benchmark between chaotropes and kosmotropes. These results suggested a quantitative measurement of kosmotropicity/chaotropicity, where the anion would be kosmotropic if its Δδ_H2O were higher than that of Cl^- and chaotropic for the opposing condition. Moreover, the results showed that the effects of the cations on the water structure were minimal, which was consistent with minimal charge transfer between the cations and water. The overall results of this study suggest that the Hofmeister ion effect is a global effect, while local interactions of ions with solutes also play a key role.     

Surprising Hofmeister Effects on the Bending Vibration of Water

The Hofmeister series, which originally described the specific ion effects on the solubility of macromolecules in aqueous solutions, has been a long‐standing unsolved and exceptionally challenging mystery in chemistry. The complexity of specific ion effects has prevented a unified theory from emerging. Accumulating research has suggested that the interactions among ions, water and various solutes play roles. However, among these interactions, the binding between ions and solutes is receiving most of the attention, whereas the effects of ions on the hydrogen‐bond structure in liquid water have been deemed to be negligible. In this study, attenuated‐total‐reflectance Fourier transform infrared spectroscopy is used to study the infrared spectra of salt solutions. The results show that the red‐ and blue‐shifts of the water bending band are in excellent agreement with the characteristic Hofmeister series, which suggests that the ions’ effects on water structure might be the key role in the Hofmeister phenomenon.     

Hofmeister series reversal for lysozyme by change in pH and salt concentration: insights from electrophoretic mobility measurements

Hofmeister series reversal can occur with change in pH, or increase in salt concentration. The phenomena are a challenge for any theory of ion specific effects. Recent theoretical work predicts how a complex interplay between ionic sizes, hydration and dispersion forces explains Hofmeister series reversal. Electrophoretic mobility measurements on lysozyme suspensions reported here are consistent with the theory.     

On the mechanism of the hofmeister effect

Vibrational sum frequency spectroscopy was used to probe fatty amine monolayers spread on various electrolyte solutions. The spectra revealed ion specific changes in both monolayer ordering and water structure with the former following the Hofmeister series. Separate measurements of the surface potential as a function of ion tracked closely to changes in alkyl chain structure, but less closely to changes in water structure. The disruption of the monolayer ordering could be ascribed to the relative ability of the ions to penetrate past the hydrophilic surface of the monolayer's headgroups and into the more hydrophobic portion of the thin film. The corresponding trends observed in the surface water structure showed significant deviations from the Hofmeister series, leading to the conclusion that the changes in surface water structure, often credited with being the origin of Hofmeister effects, are probably not of primary importance. On the other hand, dispersion forces almost certainly play a large role in the order of the Hofmeister series.     

Not only pH. Specific buffer effects in biological systems

The aim of this work is to overview the specific effect of pH buffers in biological systems. The pH of a buffer solution changes only slightly when a small amount of a strong acid or bases is added to it. This is widely accepted and applied both in chemical and in biological (i.e. enzyme catalysis) systems. Here we show some examples – spanning from pH measurements, enzyme activities, electrophoretic mobilities, antibody aggregation, protein thermal stability – that demonstrate additional roles of buffers. They not only set pH, but also address specific ion effects, in terms of Hofmeister series, when strong electrolytes are also added. From the experimental data referred to some charged biological moieties it emerges that different buffers, at the same nominal pH, can specifically adsorb at the charged surface. Buffer specific adsorption modifies several molecular and macroscopic properties amongst which electrophoretic mobilities, and hence effective surface charges, are particularly significant. More importantly, buffers' weak electrolytes, even at low concentration, are found to compete for the adsorption at the charged surfaces with strong electrolytes, thus modulating Hofmeister effects.     

The interfacial tension concept,as revealed by fluctuations

A simple, didactic model that could have conclusively interpreted the complexity of specific salt (Hofmeister-) effects on protein solubility and function, using a single physical quantity as a central parameter, has long been missing. Via surveying a row of recent papers we show in this review that a phenomenological formalism based on the salt-induced change of protein–water interfacial tension (∆γ) is able to account for a wide range of Hofmeister effects, including also such “exceptions”, where inverse or “V-shaped” Hofmeister series occurs. A close relationship between protein–water interfacial tension and conformational fluctuations is pinpointed on theoretical grounds, then it is shown how one can use a complex experimental arsenal to demonstrate conformational fluctuations on two prototypical proteins, the membrane protein bacteriorhodopsin and the cytoplasmic protein myoglobin. Finally, via the results of recent and new molecular dynamics simulations on a model peptide, the tryptophan-cage miniprotein, independent evidences are given in favor of the interfacial tension concept, at the same time demonstrating the predictive power of the theory. It is shown that salt-induced fluctuation changes of surface-exposed amino acid groups can be used as a sensitive measure for mapping the local features of Hofmeister effects on protein conformations. General implications of the interfacial tension concept are also discussed.     

Ion specific surface forces between membrane surfaces

Entities such as ion distributions and forces between lipid membranes depend on effects due to the intervening salt solution that have not been recognized previously. These specific ion or Hofmeister effects influence membrane fusion. A typical illustrative example is this: measurements of forces between double-chained cationic bilayers adsorbed onto molecularly smooth mica surfaces across different 0.6-2 mM salt solutions have revealed a large degree of ion specificity [Pashley et al. J. Phys. Chem. 1986, 90, 1637]. This has been interpreted in terms of very specific anion "binding" to the adsorbed bilayers, as it would too for micelles and other self-assembled systems. However, we show here that inclusion of nonelectrostatic (NES) or ionic dispersion potentials acting between ions and the two surfaces explains such "ion binding". The observed Hofmeister sequence for the calculated pressure without any direct ion binding is given correctly. This demonstrates the importance of a source of ion specificity that has been ignored. It is due to ionic physisorption caused by attractive NES ionic dispersion potentials. There appear to be some far reaching consequences for interpretations of membrane intermolecular interactions in salt solutions.     

Ions at interfaces: the central role of hydration and hydrophobicity

It is increasingly being accepted that solvation properties of ions and interfaces (hydration of ions, hydrophobic or hydrophilic character of interfaces) play a fundamental role in ion-surface interaction in water. However, a fundamental understanding of the precise role of solvation in ionic specificity in colloidal systems is still missing, although important progress has been made over the last years. We present in this contribution experimental evidences (including also ions not usually included in specific ion studies) together with Molecular Dynamics (MD) simulations that highlight the importance of the hydration of ions and surfaces in order to understand the origin of ionic specificity. We first show that both surface polarity and ion hydration determine the sorting of ions according to their ability to induce specific effects (the so-called Hofmeister series). We extend these classical series by considering the addition of the inorganic anions IO₃⁻, BrO₃⁻ and ClO₃⁻, which present unusual properties as compared with the ions considered in classical Hofmeister series. We also consider big hydrophobic organic ions such as tetraphenylborate anion (Ph₄B⁻) and tetraphenylarsonium cation (Ph₄As⁺) that in the context of the Hofmeister series behave as super-chaotropes ions.     

Hydrophobic and Hofmeister effects on the adhesion of spider silk proteins onto solid substrates: an AFM-based single-molecule study

AFM-based single-molecule force spectroscopy has been used to study the effect of Hofmeister salts and protein hydrophobicity on the adhesion of recombinant spider silk proteins onto solid substrates. Therefore, a molecular probe consisting of a spider silk protein and an AFM tip has been developed, which (i) is a well-defined, small system that can be simulated by molecular dynamics simulations, (ii) allows access to the whole soluble concentration range for ions, and (iii) provides the distribution of desorption forces rather than just ensemble-averaged mean values. The measured desorption forces follow the Hofmeister series for anions (H2PO4-, Cl-, I-) with a stabilizing energy of more than 15 kBT for 5 M NaH2PO4. Moreover, this effect is influenced by the hydrophobicity of the spider silk protein, indicating that hydrophobic and Hofmeister effects are closely related.     

Hofmeister effect on the interfacial dynamics of single polymer molecules

The Hofmeister effect on interfacial dynamics has been discovered for single charged polymer molecules (sodium polystyrene sulfonate) adsorbed on a hydrophobic surface from an aqueous solution. The presence of ions in the aqueous solution affects the surface diffusivity, and its amplitudes and the surface friction follow the Hofmeister series-the kosmotropic ions slowed down the surface diffusivity and the chaotropic ions speeded it up. The amplitude of the surface friction exhibits a good correlation with the surface tension increment, indicating the interfacial feature of the Hofmeister effect.     

Hofmeister effects: interplay of hydration, nonelectrostatic potentials, and ion size

The classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory of colloids, and corresponding theories of electrolytes, are unable to explain ion specific forces between colloidal particles quantitatively. The same is true generally, for surfactant aggregates, lipids, proteins, for zeta and membrane potentials and in adsorption phenomena. Even with fitting parameters the theory is not predictive. The classical theories of interactions begin with continuum solvent electrostatic (double layer) forces. Extensions to include surface hydration are taken care of with concepts like inner and outer Helmholtz planes, and "dressed" ion sizes. The opposing quantum mechanical attractive forces (variously termed van der Waals, Hamaker, Lifshitz, dispersion, nonelectrostatic forces) are treated separately from electrostatic forces. The ansatz that separates electrostatic and quantum forces can be shown to be thermodynamically inconsistent. Hofmeister or specific ion effects usually show up above ≈10(-2) molar salt. Parameters to accommodate these in terms of hydration and ion size had to be invoked, specific to each case. Ionic dispersion forces, between ions and solvent, for ion-ion and ion-surface interactions are not explicit in classical theories that use "effective" potentials. It can be shown that the missing ionic quantum fluctuation forces have a large role to play in specific ion effects, and in hydration. In a consistent predictive theory they have to be included at the same level as the nonlinear electrostatic forces that form the skeletal framework of standard theory. This poses a challenge. The challenges go further than academic theory and have implications for the interpretation and meaning of concepts like pH, buffers and membrane potentials, and for their experimental interpretation. In this article we overview recent quantitative developments in our evolving understanding of the theoretical origins of specific ion, or Hofmeister effects. These are demonstrated through an analysis that incorporates nonelectrostatic ion-surface and ion-ion dispersion interactions. This is based on ab initio ionic polarisabilities, and finite ion sizes quantified through recent ab initio work. We underline the central role of ionic polarisabilities and of ion size in the nonelectrostatic interactions that involve ions, solvent molecules and interfaces. Examples of mechanisms through which they operate are discussed in detail. An ab initio hydration model that accounts for polarisabilities of the tightly held hydration shell of "cosmotropic" ions is introduced. It is shown how Hofmeister effects depend on an interplay between specific surface chemistry, surface charge density, pH, buffer, and counterion with polarisabilities and ion size. We also discuss how the most recent theories on surface hydration combined with hydrated nonelectrostatic potentials may predict experimental zeta potentials and hydration forces.     

Hofmeister effects on the colloidal stability of an IgG-coated polystyrene latex

The effect of various ions related to the Hofmeister series (HS) on different properties of a cationic latex covered with a protein (IgG) is analyzed in this study. NaNO3, NH4NO3, and Ca(NO3)2 were used to compare the specificity of the cations, and NaCl, NaSCN, NaNO3, and Na2SO4, to compare the specificity of the anions. Two pH values, 4 and 10, were chosen to analyze the behavior of these ions acting as counter- and co-ions. At pH 4, the total surface charge is positive, whereas at pH 10 it is negative. Three different phenomena have been studied in the presence of these Hofmeister ions: (1) colloidal aggregation, (2) electrophoretic mobility, and (3) colloidal restabilization. The specific effect of the ions was clearly observed in all experiments, obtaining ion sequences ordered according to their specificity. The most important parameter for ion ordering was the sign of the charge of the colloidal particle. Positively charged particles displayed an ion order opposite that observed for negatively charged surfaces. Another influential factor was the hydrophobic/hydrophilic character of the particle surface. IgG-latex particle surfaces at pH 10 were more hydrophilic than those at pH 4. The SCN- ion had a peculiar specific effect on the phenomena studied (1)-(3) at pH 10. With respect to the restabilization studies at high ionic strengths, new interesting results were obtained. Whereas it is commonly known that cations may provoke colloidal restabilization in negative particles when they act as counterions, our experiments demonstrated that such restabilization is also possible with positively charged particles. Likewise, restabilization of negative surfaces induced by the specific effect of chaotropic anions (acting as co-ions) was also observed.     

Specific anion effects on enzymatic activity in nonaqueous media

The present work shows that salt anions affect the activity of Pseudomonas cepacia lipase both in aqueous and in nonaqueous media (NAM) according to a Hofmeister series. The biocatalytic assay in water was the hydrolysis of p-nitrophenyl acetate, whereas the esterification between 1-hexyl-beta-D-galactopyranoside and palmitic acid was followed in an organic solvent. The solid lipase preparations to be used in NAM were obtained through lyophilization in the presence of concentrated solutions of Hofmeister salts (Na2SO4, NaH2PO4/Na2HPO4, NaCl, NaBr, NaI, NaSCN). Salts affect enzyme activity in organic media through two mechanisms: (1) enzyme protection during lyophilization; (2) enzyme activation during the reaction. At least in our case, the latter seems to be more important than the former. The decrease of the activation energy caused by the stabilization of the transition state due to "kosmotropic" anions might be the driving force of enzyme activation. According to the most recent findings, dispersion forces may be responsible of specific anion enzyme activation/deactivation in NAM.     

Hofmeister anionic effects on hydration electric fields around water and peptide

Specific ion effects on water dynamics and local solvation structure around a peptide are important in understanding the Hofmeister series of ions and their effects on protein stability in aqueous solution. Water dynamics is essentially governed by local hydrogen-bonding interactions with surrounding water molecules producing hydration electric field on each water molecule. Here, we show that the hydration electric field on the OD bond of HOD molecule in water can be directly estimated by measuring its OD stretch infrared (IR) radiation frequency shift upon increasing ion concentration. For a variety of electrolyte solutions containing Hofmeister anions, we measured the OD stretch IR bands and estimated the hydration electric field on the OD bond to be about a hundred MV∕cm with standard deviation of tens of MV∕cm. As anion concentration increases from 1 to 6 M, the hydration electric field on the OD bond decreases by about 10%, indicating that the local H-bond network is partially broken by dissolved ions. However, the measured hydration electric fields on the OD bond and its fluctuation amplitudes for varying anions are rather independent on whether the anion is a kosmotrope or a chaotrope. To further examine the Hofmeister effects on H-bond solvation structure around a peptide bond, we examined the amide I' and II' mode frequencies of N-methylacetamide in various electrolyte D(2)O solutions. It is found that the two amide vibrational frequencies are not affected by ions, indicating that the H-bond solvation structure in the vicinity of a peptide remains the same irrespective of the concentration and character of ions. The present experimental results suggest that the Hofmeister anionic effects are not caused by direct electrostatic interactions of ions with peptide bond or water molecules in its first solvation shell. Furthermore, even though the H-bond network of water is affected by ions, thus induced change of local hydration electric field on the OD bond of HOD is not in good correlation with the well-known Hofmeister series. We anticipate that the present experimental results provide an important clue about the Hofmeister effect on protein structure and present a discussion on possible alternative mechanisms.     

Influence of salt ions on binding to molecularly imprinted polymers

Salt ions were found to have an influence on template binding to two model molecularly imprinted polymers (MIPs), targeted to penicillin G and propranolol, respectively, in water–acetonitrile mixtures. Water was detrimental to rebinding of penicillin G whereas propranolol bound in the entire water–acetonitrile range tested. In 100% aqueous solution, 3-M salt solutions augmented the binding of both templates. The effects followed the Hofmeister series with kosmotropic ions promoting the largest increase. Binding was mainly of a non-specific nature under these conditions. In acetonitrile containing low amounts of water, the specific binding to the MIPs increased with the addition of salts. Binding of penicillin G followed the Hofmeister series while an ion-exchange mechanism was observed for propranolol. The results suggest that hydration of kosmotropic ions reduces the water activity in water-poor media providing a stabilizing effect on water-sensitive MIP–template interactions. The effects were utilized to develop a procedure for molecularly imprinted solid-phase extraction (MISPE) of penicillin G from milk with a recovery of 87%.     

Thermodynamic origin of hofmeister ion effects

Quantitative interpretation and prediction of Hofmeister ion effects on protein processes, including folding and crystallization, have been elusive goals of a century of research. Here, a quantitative thermodynamic analysis, developed to treat noncoulombic interactions of solutes with biopolymer surface and recently extended to analyze the effects of Hofmeister salts on the surface tension of water, is applied to literature solubility data for small hydrocarbons and model peptides. This analysis allows us to obtain a minimum estimate of the hydration b1 (H2O A(-2)), of hydrocarbon surface and partition coefficients Kp, characterizing the distribution of salts and salt ions between this hydration water and bulk water. Assuming that Na+ and SO4(2-) ions of Na2SO4 (the salt giving the largest reduction in hydrocarbon solubility as well as the largest increase in surface tension) are fully excluded from the hydration water at hydrocarbon surface, we obtain the same b1 as for air-water surface (approximately 0.18 H2O A(-2)). Rank orders of cation and anion partition coefficients for nonpolar surface follow the Hofmeister series for protein processes, but are strongly offset for cations in the direction of exclusion (preferential hydration). By applying a coarse-grained decomposition of water accessible surface area (ASA) into nonpolar, polar amide, and other polar surface and the same hydration b1 to interpret peptide solubility increments, we determine salt partition coefficients for amide surface. These partition coefficients are separated into single-ion contributions based on the observation that both Cl- and Na+ (also K+) occupy neutral positions in the middle of the anion and cation Hofmeister series for protein folding. Independent of this assignment, we find that all cations investigated are strongly accumulated at amide surface while most anions are excluded. Cation and anion effects are independent and additive, allowing successful prediction of Hofmeister salt effects on micelle formation and other processes from structural information (ASA).