Clark D S, Bailey J E
Biochim Biophys Acta. 1984 Jul 31;788(2):181-8. doi: 10.1016/0167-4838(84)90260-7.
Several characterization methods have been used to study the deactivation in aliphatic alcohols of alpha-chymotrypsin and alpha-chymotrypsin-CNBr-Sepharose 4B conjugates. Active-site titration measurements, which were used to determine the amount of catalytically active enzyme, revealed appreciable differences between the deactivation kinetics of free and immobilized chymotrypsin. In all cases for the immobilized enzyme, the kinetics of active-enzyme disappearance differed significantly from first-order. Interestingly, the estimated intrinsic activity of immobilized chymotrypsin remaining active after different exposure times to 50% n-propanol solution increased somewhat as a result of exposure to alcohol. These findings were complemented by direct information, provided by EPR spectroscopy, on the effects of alcohols on the active-site configuration of spin-labeled chymotrypsin. EPR spectra of the free enzyme illustrated the appearance in different alcohol solutions of different enzyme forms with different active-site structures. EPR experiments also showed that denaturation of immobilized chymotrypsin was accompanied by unfolding of the active site that followed similar multi-step kinetics as the loss of active enzyme.
