Effects of thermodynamic nonideality in kinetic studies: evidence of an expanded intermediate complex in the inactivation of thrombin by antithrombin III

The technique of competitive chromogenic substrate hydrolysis is used to examine the inhibitory effects of sucrose and glycerol on the inactivation of thrombin by antithrombin III. This inhibition is attributed to the existence of a slight increase in volume/asymmetry associated with formation of the thrombin-antithrombin complex that subsequently undergoes covalent modification in an irreversible inactivation step. Partial reversal of the equilibrium step is thus considered to result from the effects of molecular crowding in the highly concentrated environment that is generated by the inclusion of these small insert solutes.