The inverse and direct Hofmeister series for lysozyme

Abstract

Anion effects on the cloud-point temperature for the liquid؊liquid phase transition of lysozyme were investigated by temperature gradient microfluidics under a dark field microscope. It was found that protein aggregation in salt solutions followed 2 distinct Hofmeister series depending on salt concentration. Namely, under low salt conditions the association of anions with the positively charged lysozyme surface dominated the process and the phase transition temperature followed an inverse Hofmeister series. This inverse series could be directly correlated to the size and hydration thermodynamics of the anions. At higher salt concentrations, the liquid-liquid phase transition displayed a direct Hofmeister series that correlated with the polarizability of the anions. A simple model was derived to take both charge screening and surface tension effects into account at the protein/water interface. Fitting the thermodynamic data to this model equation demonstrated its validity in both the high and low salt regimes. These results suggest that in general positively charged macromolecular systems should show inverse Hofmeister behavior only at relatively low salt concentrations, but revert to a direct Hofmeister series as the salt concentration is increased.liquid-liquid phase transition ͉ protein aggregation P rotein-protein interactions can lead to aggregation. Such intermolecular contacts underlie the mechanistic basis for a variety of diseases (1-4) and are key to protein crystallization (5-7). An effective way to determine the strength of biomacromolecular interactions is to study the temperature-induced phase separation that occurs in concentrated protein solutions (8). When a concentrated protein solution is cooled below its cloud-point temperature, the system can separate into 2 coexisting liquid phases: 1 rich in protein and 1 poor. As coacervate droplets of the protein-rich phase grow, the solution turns cloudy (white) as a result of the scattering of visible light. Upon standing, the solution may completely separate by gravity into a protein-rich phase and a nearly pure aqueous phase above it. The temperature at which the initial cloud point occurs provides a simple physical measurement of the forces acting among biomacromolecules. Specifically, the higher the temperature at which the initial cloud point occurs, the stronger the putative attractive forces between the protein molecules should be.Dissolved salts in aqueous solutions have a strong influence on protein-protein interactions and the subsequent aggregated states which are formed. In fact, cloud-point temperatures typically follow a specific ion order according to the Hofmeister series (5,6,(9)(10)(11)(12)(13)(14). The relative effectiveness of anions to induce protein aggregation typically follows 1 of 2 trends depending on the pH of the solution (15, 16). Above a protein's isoelectric point, the macromolecule bears a net negative charge and a direct Hofmeister series is normally observed. In this case, chaotropes such as I Ϫ , ClO 4 Ϫ , and SCN Ϫ he...

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