Effect of Buffer on Protein Stability in Aqueous Solutions: A Simple Protein Aggregation Model

Abstract

Liquid–liquid phase separation (LLPS) of proteins has recently been associated with the onset of numerous diseases. Despite several studies in this area of protein aggregation, buffer-specific effects always seem to be overlooked. In this study we investigated the influence of buffers on the phase stability of hen egg-white lysozyme (HEWL) and its respective protein–protein interactions by measuring the cloud point temperature, second virial coefficient, and interaction diffusion coefficient of several HEWL–buffer solutions (MOPS, phosphate, HEPES, cacodylate) at pH 7.0. The results indicate that the buffer molecules, depending on their hydration, adsorb on the protein surface, and modulate their electrostatic stability. The obtained information was used to extend the recently developed coarse-grained protein model to incorporate buffer-specific effects. Treated by Wertheim’s perturbation theory the model qualitatively correctly predicted the experimentally observed phase separation of all investigated HEWL–buffer solutions, and further allowed us to predict the phase stability of protein formulations even in experimentally unattainable conditions. Since the theory can be straightforwardly extended to include multiple components it presents a useful tool to study protein aggregation in crowded cell-like systems.

Influence of Low Molecular Weight Salts on the Viscosity of Aqueous-Buffer Bovine Serum Albumin Solutions

“…Since the salt ions are too small to affect the size of the protein by binding to its surface, we have assumed that the change in size is a consequence of the formation of protein aggregates. This has been observed previously in the determination of the cloud point temperature of hen’s egg white lysozyme solutions [ 34 ] and of BSA [ 23 ]. To further investigate the effect of salt ions on the aggregation tendency, we measured the zeta potential of the solutions in the presence of various salts.…”

“…The viscosity of the solution increased with increasing concentration of the added LiCl, NaCl, NaBr and NaI salts, while it decreased with the addition of KCl, RbCl and CsCl salts. Similar salt-specific trends in viscosity are also observed in protein-free salt buffer and salt water solutions [ 34 ]. Although the molar concentration of salt in the solution exceeds the molar concentration of BSA (∼1.5 mol/L) by about 10–100-fold, the mass concentrations of salt and protein are of the same order of magnitude (for example, 1 mol/L solution of LiCl is 42.4 mg/mL and of CsCl is 168.4 mg/mL; the concentration of BSA was 100 mg/mL).…”

Understanding specific ion effects and the Hofmeister series

“…A natural extension to mixed salt systems are buffered solutions, such as those relevant to SIE in biological systems. 53,175,[348][349][350] In addition to their roles in modulating pH, buffers compete with co-existing ''Hofmeister ions'' to influence, both cooperatively and independently, enzyme activities, electrophoretic mobilities, antibody aggregation and protein thermal stability. The screening and competitive behaviour of buffers has recently been modelled, showing the importance of ionic strength in this respect.…”

Interaction of Aqueous Bovine Serum Albumin with Silica Aerogel Microparticles: Sorption Induced Aggregation

“…These ion-specific interactions arise from the Hofmeister series, which is well established in protein systems [ 25 , 26 , 27 ]. The fact that the ζ-potential of silica aerogel is negative even at pH = 3.0 strengthens the idea of the specific sorption of phosphate and acetate anions form the corresponding media.…”