Pressure and concentration dependence of nucleation kinetics for crystallization of subtilisin

Nucleation kinetics of subtilisin was studied as a function of supersaturation (10–70 mg/ml) and pressure (0.1–34 MPa). The nucleation was found to have a 1.6 order dependence on supersaturation. Extent of nucleation decreased by a factor of 60 as the pressure increased from 0.1 to 34 MPa. High pressure was also used as a probe to understand the nucleation behavior of subtilisin and an estimation of the pressure-dependence of nucleation rate provided a value of 330 cm³/mol for the activation volume for nucleation. The residues in the contact region were determined to be hydrophilic by protein structure analysis. Responsible mechanisms are hypothesized.