Ion-specific effects at the protein surface are investigated here in light of the changes they infer to surface water dynamics, as observed by ¹H NMR relaxation (at 20 MHz). Two well-known proteins, hen egg-white lysozyme (LZM) and bovine serum albumin (BSA), show qualitatively opposite trends in the transverse relaxation rate, R₂(¹H), along a series of different monovalent salt anions in the solution. Presence of salt ions increases R₂(¹H) in the case of lysozyme and diminishes it in the case of BSA. The effect magnifies for larger and more polarizable ions. The same contrasting effect between the two proteins is observed for protein–solvent proton exchange. This hints at subtle effects ion-binding might have on the accessibility of water surface sites on the protein. We suggest that the combination of the density of surface charge residues and surface roughness, at the atomic scale, dictates the response to the presence of salt ions and is proper to each protein. Further, a dramatic increase in R₂(¹H) is found to correlate closely with the formation of protein aggregates. The same ordering of salts in their ability to aggregate lysozyme, as seen previously by cloud point measurements, is reproduced here by R₂(¹H). ¹H NMR relaxation data is supplemented by ³⁵Cl and ¹⁴N NMR relaxation for selected salt ions to probe the ion-binding itself.

Pharmaceutical design of protein formulations aims at maximum efficiency (protein concentration) and minimum viscosity. Therefore, it is important to know the nature of protein-protein interactions and their influence on viscosity. In this work, we investigated the dependence of the viscosity of BSA in an aqueous 20 mM acetate buffer at pH = 4.3 on protein concentration and on temperature (5–45 °C). The viscosity of the solution increased with protein concentration and was 230% higher than the viscosity of the protein-free formulation at 160 mg/mL. The viscosity decreased by almost 60% in the temperature range from 5 to 45 °C. The agreement of the modified Arrhenius theory with experiment was quantitative, whereas a hard-sphere model provided only a qualitative description of the experimental results. We also investigated the viscosity of a 100 mg/mL BSA solution as a function of the concentration of added low molecular weight salts (LiCl, NaCl, KCl, RbCl, CsCl, NaBr, NaI) in the range of salt concentrations up to 1.75 mol/L. In addition, the particle size and zeta potential of BSA-salt mixtures were determined for solutions containing 0.5 mol/L salt. The trends with respect to the different anions followed a direct Hofmeister series (Cl⁻ > Br⁻ > I⁻), whereas for cations in the case of viscosity the indirect Hofmeister series was observed (Li⁺ > Na⁺ > K⁺ > Rb⁺ > Cs⁺), but the values of particle sizes and zeta potential did not show cation-specific effects. Since the protein is positively charged at pH = 4.3, anions are more attracted to the protein surface and shield its charge, while the interaction with cations is less pronounced. We hypothesize that salt surface charge shielding reduces protein colloidal stability and promotes protein aggregate formation.

The stability of bovine serum albumin (BSA) solutions against phase separation caused by cooling the system is studied under the combined influence of added poly(ethylene glycol) (PEG) and alkali halide salts in water as solvent. The phase stability of the system depends on the concentration of the added PEG and its molecular mass, the concentration of the low molecular mass electrolyte and its nature, as also on the pH of the solution. More specifically, the addition of NaCl to the BSA-PEG mixture promotes phase separation at pH = 4.0, where BSA carries the net positive charge in aqueous solution, and it increases the stability of the solution at pH=4.6, i.e., near the isoionic point of the protein. Moreover, at pH = 4.6, the cloud-point temperature decreases in the order from NaF to NaI and from LiCl to CsCl. The order of the salts at pH = 4.0 is exactly reversed: LiCl and NaF show the weakest effect on the cloud-point temperature and the strongest decrease in stability is caused by RbCl and NaNO₃. An attempt is made to correlate these observations with the free energies of hydration of the added salt ions and with the effect of adsorption of salt ions on the protein surface on the protein–protein interactions. Kosmotropic salt ions decrease the phase stability of BSA-PEG-salt solutions at pH < pI, while exactly the opposite is true at pH = pI.