CALORIMETRIC STUDIES ON SOLID ALPHA-CHYMOTRYPSIN PREPARATIONS IN AIR AND IN ORGANIC-SOLVENTS

Differential scanning calorimetry was the method to investigate the th ermostability of chymotrypsin. The transition temperature decreased by approx. 30 degrees C when the dry enzyme became highly hydrated. High degree of hydration corresponded to extensive conformational changes during protein denaturation, reflected by large enthalpy values. Sorbi tol, lyophilized together with the enzyme, caused the destabilization of the complex within the whole range of water activities. When the en zyme was equilibrated through the apolar solvent, isooctane, stabiliza tion of chymotrypsin was observed at high water activities, compared t o equilibration in air. The presence of isooctane resulted in a remark able stabilization of the chymotrypsin-sorbitol complex. A sorbitol co ncentration of 5 mmol/g of protein was prerequisite to induce stabiliz ation when equilibrated through isooctane at high water activites. The transition enthalpy increased with increasing amounts of sorbitol. Di fferent hydration isotherms were obtained for the air-equilibrated and solvent-equilibrated enzyme preparations. Increasing amounts of buffe r salts within the chymotrypsin preparation caused the enhancement of both the temperature and the enthalpy of the transition at a water act ivity 0.97. Variations on the hydration of the preparations both offer ed the explanation to the thermal stability results and supported the evidence obtained from enzyme activity studies. Generally, the catalys t whose hydration was supressed due to its environment exhibited low e nzymatic activity.