Measuring enzyme motility in organic media using novel H-D exchange methodology

A novel deuterium (H-2) NMR technique as developed for measuring the total number of deuterons exchanged by lyophilised protein samples following hydr ogen-deuterium (H-D) exchange. Using this methodology differences in the H- D exchange behaviour of the proteolytic enzyme subtilisin Carlsberg hydrate d either in air or an organic solvent were probed as a function of hydratio n. At low thermodynamic water activity (a(w)), the degree of H-D exchange i ncreased rapidly with hydration (from anhydrous to a(w) 0.22). At a(w) 0.22 , subtilisin powders hydrated in air were found to have reached an H-D exch ange level comparable to that found upon aqueous dissolution and in agreeme nt with previous studies using lysozyme. Lyophilised subtilisin hydrated in either dichloromethane (DCM) or diisopropyl ether (DIPE) showed a pattern of exchange (vs. a(w)) comparable to that found for powders hydrated in air . However, subtilisin hydrated in n-hexane showed a significant reduction i n H-D exchange at all a(w) studied. Control experiments demonstrated that t he reduction in H-D exchange observed for subtilisin in n-hexane was not a kinetic effect. This lower fever of exchange in n-hexane implies that hydra ted subtilisin Carlsberg has a lower conformational motility and more rigid protein matrix. (C) 2000 John Wiley & Sons, inc.