PH dependence of the activation parameters for chymopapain unfolding: influence of ion pairs on the kinetic stability of proteins

Abstract

We studied the irreversible thermal denaturation of chymopapain, a papain-related cysteine proteinase. It was found that this process follows simple first-order kinetics under all conditions tested. Rate constants determined by monitoring ellipticity changes at 220 or 279 nm are essentially identical, indicating that denaturation involves global unfolding of the protein. Enthalpies (Δ H ‡ ) and entropies (Δ S ‡ ) of activation for unfolding were determined at various pH values from the temperature dependence of the rate constant. In the pH range 1.1–3.0, a large variation of both Δ H ‡ and Δ S ‡ was observed. For the few proteins studied so far (lysozyme, trypsin, barnase) it is known that activation parameters for unfolding vary little with pH. It is proposed that this contrasting behavior of chymopapain originates from the numerous ion 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 analyzed in terms of differences in the protonation behavior of carboxylic groups between the transition ( TS) and native ( N) states of the protein. For this purpose, a model of independently titrating sites was assumed, which explained reasonably well the pH dependence of activation parameters, as well as the protonation properties of native chymopapain. According to these calculations, p K values of carboxyls in TS are shifted 0.6–0.9 units upwards with respect to those in N. In addition, some groups in TS appear to be protonated with unusually large enthalpy changes.