Solvent effects on the growth kinetics of subtilisin crystals

The effects of salts on subtilisin crystallization were investigated. Three salts—NaCl, NaNO₃ and NaSCN—were selected to study the effects of different anions on growth kinetics of three subtilisin mutants—Properase^®, Purafect^® and Purafect^®OX. The effectiveness of salts in decreasing the solubility of Properase^® and Purafect^® subtilisin followed the reverse order of the Hofmeister series: SCN⁻>NO₃⁻>Cl⁻. The average length and diameter of crystals were measured during crystallization. The nature of salt changed the length/diameter ratio of crystals, indicating the changes in the relative growth rate of different crystal faces. The required supersaturation, (c−s)/s, for a given growth rate increased in the order of NaCl, NaNO₃ and NaSCN. The observed trend in required supersaturation indicates a kinetic effect and was counter to the trend for the solubility data. A rationale is provided based on the influence of ion binding and kinetics on the energetics of crystal growth and growth rate is correlated to the molar Gibbs free energy of hydration of the anion.