EFFECT OF SECONDARY STRUCTURE ON THE ACTIVITY OF ENZYMES SUSPENDED INORGANIC-SOLVENTS

Despite the extensive use and study of enzymes suspended in organic so lvents, whether activity differences between different preparations ca n be accounted for by differences in protein secondary structure is st ill unknown. To address this issue, in the current study two model enz ymes, alpha-chymotrypsin and subtilisin Carlsberg, were lyophilized an d suspended in both polar and nonpolar organic solvents. The secondary structures of the proteins in the initial aqueous solution, in the ly ophilized powder, and in the subsequent suspensions in organic solvent s were determined using infrared spectroscopy. Lyophilization perturbe d the secondary structure of both enzymes. With alpha-chymotrypsin, ly ophilization from buffer followed by suspension in ethanol, hexane, or pyridine did not alter the unfolded structure observed in the dried p owder. In contrast, with subtilisin Carlsberg, suspension of the dried enzyme in ethanol led to further perturbation of structure, whereas i n hexane, and more so in pyridine, there was some return toward native structure. Lyophilization of the aqueous protein solutions in the pre sence of either trehalose or sorbitol led to retention of more native- like structure of both enzymes in the dried solid. However, large stru ctural perturbations arose when these samples were suspended in organi c solvents. The only exception was the subtilisin-trehalose mixture, w hich regained some native structure in ethanol and hexane. The greates t changes were noted in samples suspended in pyridine, in which the in frared spectra indicated extensive intermolecular beta-sheet formation from protein aggregates. There was not any consistent correlation bet ween activity in organic solvents and either the initial structure obt ained in the dried powders or the final structure when suspended in or ganic solvents. Nor could differences in residual water contents in dr ied samples or the total water content in the organic solvent reaction system account for the activity differences. (C) 1996 Academic Press, Inc.