Solvent effects on the second virial coefficient of subtilisin and solubility

Protein-protein interactions of Properase and Purafect subtilisin in solution have been studied by static light scattering as a function of salt type (NaCl, NaSCN) and salt concentration. The second virial coefficient is a measure of two-body protein-protein interactions. The measured second virial coefficients decreased from positive to negative values with increasing salt concentration, showing that protein-protein interaction changed from repulsion to attraction. The second virial coefficient of Properase in NaSCN is lower than that in NaCl, indicating that aggregation is more favorable in NaSCN solution. Moreover, solubility was quantitatively correlated with the second virial coefficient using a theoretically based correlation. To relate the protein interactions to mechanistic contributions, the experimental osmotic second virial coefficients were fitted to a DLVO-type model, consisting of hard sphere, charge-charge electrostatic, dispersion and osmotic attraction potentials, leaving the Hamaker constant as the adjustable parameter. The fitted Hamaker constants for the two subtilisin mutants ranged from 4.4 to 6.8 kT in these salt solutions. Contributions of electrostatic, van der Waals (dispersion), and osmotic attraction potentials were compared for different salt solutions.