Additivity of the Specific Effects of Additives on Protein Phase Behavior

Protein phase behavior and protein-protein interactions can be tuned by additives. We experimentally determined the phase behavior of lysozyme solutions, namely, the cloud-point temperature (CPT), in the presence of two additives, sodium chloride (NaCl) and guanidine hydrochloride (GuHCl). Their concentrations are chosen to maintain the secondary structure, as verified by CD spectroscopy. Our data indicate that the salts affect the CPT through electrostatic screening and salt-specific contributions. At high salt concentrations, the CPT is a linear function of the additive concentration for the salts NaCl and GuHCl as well as for a nonionic additive, glycerol, and a solvent, dimethyl sulfoxide (DMSO). Their molar temperature increments, which rank their specific effects on the CPT (NaCl > 0 > DMSO > glycerol > GuHCl), are found to be essentially independent of the protein concentration. In particular, the specific effects of NaCl and GuHCl in mixtures are found to be additive, indicating the absence of synergies or suppressions between both salts. Thus, molar temperature increments represent a characteristic measure for the specific effects of additives on protein interactions, which is easily accessible in lab experiments and which will help to characterize the effects of additives on protein interactions and phase behavior.