Using phase transitions to investigate the effect of salts on protein interactions

We have investigated liquid-liquid and solid-liquid phase separation of aqueous solutions of lysozyme. We have determined experimentally how the phase transition temperatures depend on protein concentration and the ionic composition of the solution. For a wide range of solution conditions, we find that the cloud-point temperature T-cloud-which signals the onset of liquid-liquid phase separation-is 15-45 degrees C below the crystallization temperature T-xtal. This indicates that liquid-liquid phase separation occurs in a highly metastable solution. When a series of chloride, bromide, and sulfate salts are added to lysozyme, we find that T-cloud varies by as much as 60 degrees C over the salt concentration range of 0.2M to 1.5M. The precise change in T-cloud depends sensitively on the identities of both the cation and the anion of the added salt. The effect of salts on T-xtal is very similar to their effect on T-cloud. The Derjaguin-Landau-Verwey-Overbeek theory for the interaction energy between charged spheres cannot account for our observations and indicates that hydration forces play an important role in protein interactions.