PH-DEPENDENCE OF THE ACTIVATION PARAMETERS FOR CHYMOPAPAIN UNFOLDING - INFLUENCE OF ION-PAIRS ON THE KINETIC STABILITY OF PROTEINS

We studied the irreversible thermal denaturation of chymopapain, a pap ain-related cysteine proteinase. It was found that this process follow s simple first-order kinetics under all conditions tested. Rate consta nts determined by monitoring ellipticity changes at 220 or 279 nm are essentially identical, indicating that denaturation involves global un folding of the protein. Enthalpies (Delta H double dagger) and entropi es (Delta S double dagger) of activation for unfolding were determined at various pH values from the temperature dependence of the rate cons tant. In the pH range 1.1-3.0, a large variation of both Delta H doubl e dagger and Delta S double dagger was observed. For the few proteins studied so far (lysozyme, trypsin, barnase) it is known that activatio n parameters for unfolding vary little with pH. It is proposed that th is contrasting behavior of chymopapain originates from the numerous io n pairs - especially those with low solvent accessibilities - present in its molecular structure. In contrast, fewer, more exposed ion pairs are present in the other proteins mentioned above. Our results were a nalyzed in terms of differences in the protonation behavior of carboxy lic groups between the transition (TS) and native (N) states of the pr otein. For this purpose, a model of independently titrating sites was assumed, which explained reasonably well the pH dependence of activati on parameters, as well as the protonation properties of native chymopa pain. According to these calculations, pK values of carboxyls in TS ar e shifted 0.6-0.9 units upwards with respect to those in N. In additio n, some groups in TS appear to be protonated with unusually large enth alpy changes. (C) 1998 Elsevier Science B.V. All rights reserved.