Solvent role in protein crystallization as determined by pressure dependence of nucleation rate and solubility

The effects of pressure on the nucleation rate provide information about the transition state during crystal nucleation, providing insight into solvent roles in nucleation. Nucleation kinetics and equilibrium solubilities of Properase subtilisin were measured at four pressures: 0.1, 6.8, 13.6, and 34 MPa for nucleation; 0.1, 13.6, 34, and 68 MPa for solubility. The nucleation rate varied with supersaturation to the 1.30 power over this range; crystal morphology was unchanged. Assuming Le'Chatelier's law to be applicable to the kinetic rate equation used to model the nucleation kinetics, the activation volume for nucleation was estimated to be 226 cm(3)/mol. The solubility dependence on pressure corresponded to a 37 cm(3)/mol overall volume change for crystallization. These results could be attributed largely to the volume change resulting from movement of water from the hydration layers to the bulk.