A TEST FOR THE 2-STAGE THERMOINACTIVATION MODEL FOR CHYMOTRYPSIN

Chymotrypsin was irreversibly inactivated at 30-130-degrees-C, pH 1.0- 7.0 and in the presence of 0-4.0 m guanidine hydrochloride (GnHCl). Th e activation enthalpy for enzyme thermoinactivation (DELTAH#) at moder ate temperatures, pH 4.0-7.0 and in less-than-or-equal-to 2 m GnHCl wa s 175-322 kJ mol-1. The activation entropy (DELTAS#) was 244-734 J mol -1 K-1. Such results are compatible with enzyme unfolding being the ra te-determining step for the thermoinactivation of native chymotrypsin. For chymotrypsin pre-unfolded at low pH, high temperature and/or in G n/HCl, DELTAH# was 30-40 kJ mol-1 and DELTAS# was between -182 and -19 1 J mol-1 K-1. Therefore, thermoinactivation of pre-unfolded chymotryp sin is likely to involve covalent bond lysis as the rate-determining s tep. A biphasic Arrhenius plot was obtained for chymotrypsin thermoina ctivation in 1.0-1.5 m GnHCl. Taken together, these results provide st rong support for the two-stage model for enzyme thermoinactivation.