Interactions in protein solutions close to liquid-liquid phase separation: ethanol reduces attractions via changes of...

Ethanol is a common protein crystallization agent, precipitant, and denaturant, but also alters the dielectric properties of solutions. While ethanol-induced unfolding is largely ascribed to its hydrophobic parts, its effect on protein phase separation and inter-protein interactions remains poorly understood. Here, the effects of ethanol and NaCl on the phase behavior and interactions of protein solutions are studied in terms of the metastable liquid-liquid phase separation (LLPS) and the second virial coefficient B₂ using lysozyme solutions. Determination of the phase diagrams shows that the cloud-point temperatures are reduced and raised by the addition of ethanol and salt, respectively. The observed trends can be explained using the extended law of corresponding states as changes of B₂. The results for B₂ agree quantitatively with those of static light scattering and small-angle X-ray scattering experiments. Furthermore, B₂ values calculated based on inter-protein interactions described by the Derjaguin-Landau-Verwey-Overbeek (DLVO) potential and considering the dielectric solution properties and electrostatic screening due to the ethanol and salt content quantitatively agree with the experimentally observed B₂ values.