Implications of specific ion effects on salt-induced proteome precipitation in organic solvent.

Maximizing recovery of water-soluble proteins through acetone precipitation

Specific ion effects ranking in the Hofmeister sequence are ubiquitous in biochemical, industrial, and atmospheric processes. In this experimental study specific ion effects inexplicable by the classical DLVO theory have been investigated at curved water-metal interfaces of gold nanoparticles synthesized by a laser ablation process in liquid in the absence of any organic stabilizers. Notably, ion-specific differences in colloidal stability occurred in the Hückel regime at extraordinarily low salinities below 50 μM, and indications of a direct influence of ion-specific effects on the nanoparticle formation process are found. UV-vis, zeta potential, and XPS measurements help to elucidate coagulation properties, electrokinetic potential, and the oxidation state of pristine gold nanoparticles. The results clearly demonstrate that stabilization of ligand-free gold nanoparticles scales proportionally with polarizability and antiproportionally with hydration of anions located at defined positions in a direct Hofmeister sequence of anions. These specific ion effects might be due to the adsorption of chaotropic anions (Br(-), SCN(-), or I(-)) at the gold/water interface, leading to repulsive interactions between the partially oxidized gold particles during the nanoparticle formation process. On the other hand, kosmotropic anions (F(-) or SO4(2-)) seem to destabilize the gold colloid, whereas Cl(-) and NO3(-) give rise to an intermediate stability. Quantification of surface charge density indicated that particle stabilization is dominated by ion adsorption and not by surface oxidation. Fundamental insights into specific ion effects on ligand-free aqueous gold nanoparticles beyond purely electrostatic interactions are of paramount importance in biomedical or catalytic applications, since colloidal stability appears to depend greatly on the type of salt rather than on the amount.

Biophysical studies in the last two decades have clearly demonstrated that salts affect biomolecules in an ion-specific manner (i. e., Hofmeister Effects). Studies performed upon such diverse biological processes such as protein folding, protein precipitation, protein coacervation and phase separation, and protein oligomerization, have all shown that this ion specificity is directly related to how individual ions interact with biomolecular surfaces. Interestingly, although ion-specific effects upon enzyme catalytic processes are well-known in the literature, a molecular level description of these effects has not yet been made available. For example, it is not clear whether ion-specific effects observed in enzyme catalysis are directly related to how ions modulate the enzyme's folding free energy, or not. This work attempts to address this need by investigating ion-specific effects upon the enzymatic activity and folding free energy of a well-characterized enzyme system, Ribonuclease A (RNase A). To this end we have developed a robust framework to analyze and quantify ion-specific effects upon the RNase A catalyzed phosphate ring opening reaction of cCMP (Cytidine 2':3'-cyclic monophosphate monosodium salt). Our studies show that both the folding thermodynamics and the Michaelis-Menten kinetic parameters of this enzyme show ion-specific salt dependence. However, even through salt addition affects the folding free energy and enzyme catalysis of RNase A in an ion-specific manner, these effects are not necessarily directly related to each other. Ion-specific effects observed in protein folding reflects mostly how an individual ion interacts with the overall protein surface; while alternatively, ion-specific effects on enzyme activity indicate how a given ion interacts with the enzyme active site surface or alternatively, how ions interact with the substrate molecule as represented by changes in the substrate thermodynamic activity coefficient.

In this study, the enantioselective analysis of fluoxetine and norfluoxetine in plasma samples was performed by the protein precipitation method and high performance liquid chromatography with fluorescence detection (PP/LC-FD). Different precipitating agents - organic solvents, acids, and salts - in several proportions were available. The Bradford colorimetric method employed for evaluation of the efficiency of protein precipitation, has shown that for the sake of simplicity and percentage of protein precipitation (99.7%), acetonitrile was most effective when added at a ratio of 3:1 (acetonitrile/plasma, v/v). The quantification limit of the PP/LC-FD method was 30 ng mL-1 for the four enantiomers. The response of the proposed method was linear over a dynamic range from LOQ to 1000 ng mL-1, with correlation coefficients higher than 0.9973. In conclusion, the PP/LC-FD method can be successfully used to analyze plasma samples from ageing patients undergoing therapy with fluoxetine.

Salt solubility in organic solvents is of particular interest in industry, for example, for carbon capture and storage or utilization processes, battery technology, or biotechnology. Electrolyte thermodynamic models have been developed to reduce the experimental effort for the design of an electrolyte toward desired properties, for example, high solubility in an organic solvent. In this work, the solubility of nine salts (NaCl, KCl, CsCl, NaHCO3, KHCO3, CsHCO3, Na2CO3, K2CO3, and Cs2CO3) in the organic solvents methanol, ethanol, and N-methyl-2-pyrrolidone (NMP) was studied at temperatures between 288.15 and 348.15 K. These systems were chosen since the largest amount of experimental data points were available in order to ensure a broad set of data for high modeling accuracy. Experimental solubility data were collected from literature, and missing data were measured in this work by both ion-chromatography analysis and the all-gravimetric method. The thermodynamic solubility product KSP of the salts was determined at 298.15 K and 1 bar. These KSP values do NOT depend on the solvent; that is, once known, they can be used to predict the solubility in any solvent or solvent mixture. KSP requires that the solid form of the precipitating salt be the same in different organic solvents. Therefore, powder X-ray diffractometer measurements were carried out to investigate possible hydrate formation or solvate formation, and Karl Fischer measurements were used to validate the absence of water. The equation of state ePC-SAFT was applied to model salt solubility in organic solvents by accounting for concentration-dependent dielectric constants within Debye–Hückel theory and Born theory. The required KSP values of the salts were determined using experimental literature data on the salt solubility in water together with the corresponding mean ionic activity coefficients (MIACs) at saturation. The availability of KSP and the predicted MIACs allowed modeling of the salt solubility in organic solvents in excellent agreement with experimental data. Furthermore, solvent-specific analysis and ion-specific analysis revealed a non-intuitive behavior of salt solubility in the organic solvents. One example is that ion-specific effects of salt solubility in alcohols are not valid in other organic solvents such as NMP. Furthermore, in contrast to aqueous solutions, salt solubility in organic solvents does not depend linearly on the cation size. Thus, experimental rules of thumb cannot be applied, and the experimental effort to screen salt solubility in organic solvents can only be significantly reduced by theoretical approaches such as ePC-SAFT.

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Molecular chaperones are a diverse group of proteins that interact with partially folded protein states to stabilize and prevent their mutual (illicit) association. Proteins require involvement with molecular chaperones throughout their lifespan: from their synthesis and folding through intracellular transport, membrane translocation, and to their ultimate degradation. Small heat-shock proteins (sHsps) are a ubiquitous family of molecular chaperones that are found in all organisms. Unlike many of the well-characterized chaperones, for example from the Hsp60 and Hsp70 families, sHsps are not involved in regulating protein folding. Instead, under conditions of cellular stress, such as elevated temperatures, they interact and stabilize partially folded target proteins to prevent their aggregation and precipitation. Because of this ability, their expression is elevated in many protein diseases that are characterized by protein aggregation and precipitation, including Alzheimer's, Creutzfeldt–Jakob, and Parkinson's diseases. The principal lens protein, a-crystallin, is a sHsp. Its chaperone ability is important in preventing lens protein precipitation and hence in maintaining lens transparency. This review summarizes the salient structural features of sHsps that enable them to act as highly efficient chaperones to prevent protein precipitation under stress conditions. The mechanism of chaperone action and the state of the target protein when interacting with sHsps are also discussed. Finally, diseases in which sHsp expression is elevated are discussed including the potential roles of sHsps and their therapeutic uses in the treatment of these diseases.

Proteins in ionic liquids (ILs) and deep eutectic solvents (DESs) have gained significant attention due to their potential applications in various fields, including biocatalysis, bioseparation, biomolecular delivery, and structural biology. Scattering approaches including dynamic light scattering (DLS) and small-angle X-ray and neutron scattering (SAXS and SANS) have been used to understand the solution behavior of proteins at the nanoscale and microscale. This review provides a thorough exploration of the application of these scattering techniques to elucidate protein properties in ILs and DESs. Specifically, the review begins with the theoretical foundations of the relevant scattering approaches and describes the essential solvent properties of ILs and DESs linked to scattering such as refractive index, scattering length density, ion-pairs, liquid nanostructure, solvent aggregation, and specific ion effects. Next, a detailed introduction is provided on protein properties such as type, concentration, size, flexibility and structure as observed through scattering methodologies. This is followed by a review of the literature on the use of scattering for proteins in ILs and DESs. It is highlighted that enhanced data analysis and modeling tools are necessary for assessing protein flexibility and structure, and for understanding protein hydration, aggregation and specific ion effects. It is also noted that complementary approaches are recommended for comprehensively understanding the behavior of proteins in solution due to the complex interplay of factors, including ion-binding, dynamic hydration, intermolecular interactions, and specific ion effects. Finally, the challenges and potential research directions for this field are proposed, including experimental design, data analysis approaches, and supporting methods to obtain fundamental understandings of complex protein behavior and protein systems in solution. We envisage that this review will support further studies of protein interface science, and in particular studies on solvent and ion effects on proteins.

Self-assembled multilayers of a strong polyanion, poly(sodium 4-styrenesulfonate) (PSS), and a strong polycation, poly[(diallyl-dimethyl-ammonium chloride)-stat-(N-methyl-N-vinyl acetamide)] (P(DADMAC-stat-NMVA)), are fabricated on silicon substrates. This article addresses the effect of electrostatics versus ion specificity. Therefore, multilayer formation and growth are investigated as a function of the charge density of the polycation, the type of salt in the polyelectrolyte dipping solution, and its ionic strength. This study focuses on monovalent ions (Li(+), Na(+), K(+), Cs(+), Rb(+), F(-), Cl(-), Br(-), and ClO(3)(-)). Ellipsometry and X-ray reflectometry data indicate that anions have a significantly larger effect on the thickness of the multilayer, but contrary to other studies on ion-specific effects, the influence of the type of cation is not negligible at higher salt concentrations. Larger ions, with smaller hydration shells, are highly polarizable and consequently interact strongly with charged polyelectrolytes, resulting in thicker and rougher multilayers. AFM studies confirm a higher roughness of the multilayer prepared from larger anions. The substrate can mask ion-specific effects over a distance of about 10 nm. Ion-specific effects become important above an ionic strength of 0.1 M in the case of anions and above an ionic strength of 0.25 M for cations. At lower ionic strengths, electrostatic interactions between and within the polyelectrolyte chains are dominating. Reducing the degree of polymer charge down to 75% does not shift this threshold of ionic strength. It is shown that a combination of ionic strength, polymer charge, and type of ion is a suitable tool for tuning the mobility and stability of polyelectrolyte multilayers.

The interaction of nanoparticles with cell membranes is critical to understand and control the structural change and molecular transport of cell membranes for medicines and medical diagnostics, in which hydrophobic interaction is often involved. We examine the specific ion effect on the interaction of semihydrophobic nanoparticle with zwitterionic phospholipid bilayer in aqueous media added with different types of salts. Specifically, we compare the effect of different anions or cations on the adsorption of carboxyl-functionalized polystyrene nanoparticle on supported lipid bilayer and its induced bilayer disruption. By adding different anions at the same ionic concentration to the nanoparticle-lipid bilayer interface, we observe that the growth rate of nanoparticle-induced lipid-poor regions follows the exact Hofmeister anion order of CH3COO(-) > Cl(-) > NO3(-) ≫ SCN(-), suggesting the regulated hydrophobic interaction by anions. In contrast, the specific cation effect on nanoparticle-induced disruption rate of lipid bilayer does not follow the Hofmeister cation order and instead exhibits a trend of Cs(+) ∼ Rb(+) > Na(+) ≫ N(CH3)4(+). It is suggested that the effect of specific ions can be exploited as a simple and efficient approach to modify the nanoparticles-biomembrane interactions with the implication from drug delivery to nontoxic nanomaterial design.

Specific ion effects are of high impact in colloid science and dominate processes in aqueous systems from protein folding or precipitation to ordering of particles or macromolecules in bulk solutions. Due to the large internal interface of colloidal systems especially interfacial ion effects are of importance. This paper presents a new insight into the specific ion effects at the air/water interface of monovalent electrolyte solutions and their consequences for long-range interactions in colloidal systems. Solely, in an asymmetric film (i.e. wetting film) one can determine the sign and precise value of the surface potential of the free air/water surface. It is shown that the all over charges of the interfacial region, which are affected by the type of ion, dominate the interfacial forces even over several tens of nm. This is of interest for tailoring the stability of colloidal systems. It is clearly shown that the air/water interface is negatively charged and that both anions and cations affect the surface potential even at very low electrolyte concentrations (10−4 M).

Clusterin (CLU) is a potent extracellular chaperone that inhibits protein aggregation and precipitation otherwise caused by physical or chemical stresses (e.g. heat, reduction). This action involves CLU forming soluble high molecular weight (HMW) complexes with the client protein. Other than their unquantified large size, the physical characteristics of these complexes were previously unknown. In this study, HMW CLU-citrate synthase (CS), HMW CLU-fibrinogen (FGN), and HMW CLU-glutathione S-transferase (GST) complexes were generated in vitro, and their structures studied using size exclusion chromatography (SEC), ELISA, SDS-PAGE, dynamic light scattering (DLS), bisANS fluorescence, and circular dichroism spectrophotometry (CD). Densitometry of Coomassie Blue-stained SDS-PAGE gels indicated that all three HMW CLU-client protein complexes had an approximate mass ratio of 1:2 (CLU:client protein). SEC indicated that all three clients formed complexes with CLU>or=4x10(7) Da; however, DLS estimated HMW CLU-FGN to have a diameter of 108.57+/-18.09 nm, while HMW CLU-CS and HMW CLU-GST were smaller with estimated diameters of 51.06+/-6.87 nm and 52.61+/-7.71 nm, respectively. Measurements of bisANS fluorescence suggest that the chaperone action of CLU involves preventing the exposure to aqueous solvent of hydrophobic regions that are normally exposed by the client protein during heat-induced unfolding. CD analysis indicated that, depending on the individual client protein, CLU may interact with a variety of intermediates on protein unfolding pathways with different amounts of native secondary structure. In vivo, soluble complexes like those studied here are likely to serve as vehicles to dispose of otherwise dangerous aggregation-prone misfolded extracellular proteins.

The article shows that the type and concentration of inorganic salt can be translated into the structure of the bulk phase and the performance properties of ecological all-purpose cleaners (APC). A base APC formulation was developed. Thereafter, two types of salt (sodium chloride and magnesium chloride) were added at various concentrations to obtain different structures in the bulk phase. The salt addition resulted in the formation of spherical micelles and—upon addition of more electrolyte—of aggregates having a lamellar structure. The formulations had constant viscosities (ab. 500 mPa·s), comparable to those of commercial products. Essential physical-chemical and performance properties of the four formulations varying in salt types and concentrations were evaluated. It was found that the addition of magnesium salt resulted in more favorable characteristics due to the surface activity of the formulations, which translated into adequately high wettability of the investigated hydrophobic surfaces, and their ability to emulsify fat. A decreasing relationship was observed in foaming properties: higher salt concentrations lead to worse foaming properties and foam stability of the solutions. For the magnesium chloride composition, the effect was significantly more pronounced, as compared to the sodium chloride-based formulations. As far as safety of use is concerned, the formulations in which magnesium salt was used caused a much lesser irritation compared with the other investigated formulations. The zein value was observed to decrease with increasing concentrations of the given type of salt in the composition.

Osmotic and specific ion effects are the most frequently mentioned mechanisms by which saline substance reduces plant growth. However, the relative importance of osmotic and specific ion effect on plant growth seems to vary depending on the salt tolerance of the plant under study. Tall wheatgrass (TW), perennial ryegrass (PR), African millet (AM) and Rhodesgrass (Rh) were grown in nutrient solution with sodium chloride (NaCl), sodium sulfate (Na2SO4), potassium chloride (KCl), and potassium sulfate (K2SO4) salinity up to electrical conductivity (EC) 27 dS m−1. Growth of all plant species decreased significantly at high level (EC 27 dS m−1) of NaCl and Na2SO4 salts. However, the growth of none of the plant species was affected significantly by KCl and K2SO4 at any level. Even leaf and shoot fresh weights were enhanced by K2SO4 in all plant species, except AM. Chlorine (Cl) was taken up in similar quantities from KCl and NaCl solutions and the content of the respective cations was similar to each other. Further sensitivity to sulfate and chloride was equal when sodium concentrations in shoots were equal, regardless of the anion composition of the media. The sodium (Na) concentration of the leaves of the plant species increased with increased NaCl and Na2SO4 levels in the nutrient solutions. The leaf Na concentration of TW was lower than that of the other plant species. However, the root Na concentration of TW was higher than that of the other plant species. Increased NaCl and Na2SO4 concentrations had a marked effect on leaf water potential of all plant species, and the TW showed higher leaf water potential at all levels of salts. Tall wheatgrass adjusted osmotically by accumulating electrolytes from the nutrient solution and by accumulation of glycinebetaine. Sodium was generally found more injurious than Chloride in all the four forage species. Salt tolerance could be ascribed as greater exclusion of Na ion.

Inhibition of seed germination in alfalfa varieties Rambler, Roamer, and Beaver by iso-osmotic potentials of different substrates varied widely. Sodium and potassium sulfate and sodium chloride were most inhibitory. Chlorides of potassium and magnesium and magnesium sulfate were least effective; mannitol and polyethylene glycol were intermediate. Ion toxicity was determined by measuring germination recovery after treatment with solutions containing equivalent weights of salt. No salts were toxic at 25 meq/liter. Sodium sulfate and magnesium chloride appeared highly toxic at 200 meq/liter; other salts were less toxic, with sodium chloride showing the least effect of all. All salts were highly toxic at 400 meq/liter. Germination recovery after treatment with polyethylene glycol and mannitol was good, clearly distinguishing osmotic and specific ion effects. Variety Beaver was the most tolerant of both the osmotic and toxic effects of salt.