Abstract
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.
Highlights
Bovine serum albumin (BSA) is a protein found in bovine blood serum
In addition to electrostatic interactions, chaotropic ions (e.g., I, Cs+ ) can mediate the so-called hydrophobic interactions that cause a decrease in the viscosity of the solution. (i) We have shown the dependence of the viscosity of 100 mg/mL BSA solutions on the concentration of the added low molecular weight salts (LiCl, NaCl, KCl, RbCl, CsCl, NaBr, NaI) in the range of salt concentrations up to 1.75 mol/L at 25 °C and (ii) on the temperature in the range of 5 to 45 °C at a salt concentration of 1 M
The focus of this study was on the ion-specific effects on the viscosity of BSA solutions in acetate buffer with pH = 4.3 at different temperatures and salt concentrations
Summary
Bovine serum albumin (BSA) is a protein found in bovine blood serum. One of its main features is specific binding sites that have physiological significance for transport, modulation and inactivation of metabolite and drug activities [1,2,3,4]. Because of their high polarizability, kosmotropic ions bind the hydration water more tightly, causing protein molecules to prefer the hydrophobic protein-protein interactions in solution This effect is called salting-out and decreases the solubility of the protein. Chaotropic ions have low polarizabilities, and preferentially bind in the layer between water and protein molecules, resulting in destabilization of the protein molecules This effect is called saltingin and increases the solubility of the protein in solution [24,25,26]. (i) We have shown the dependence of the viscosity of 100 mg/mL BSA solutions on the concentration of the added low molecular weight salts (LiCl, NaCl, KCl, RbCl, CsCl, NaBr, NaI) in the range of salt concentrations up to 1.75 mol/L at 25 °C and (ii) on the temperature in the range of 5 to 45 °C at a salt concentration of 1 M. The results of our work are described and discussed in the Results and Discussion section, and the conclusions are summarized in the Conclusions section
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