Estimating the degree of expansion in the transition state for protein unfolding:: Analysis of the pH dependence of the rate constant for caricain denaturation

The thermal denaturation of caricain (the most alkaline of papain-related proteinases) was studied in acid media. Under all conditions tested, caricain denatured irreversibly following a single first-order reaction that involves simultaneous loss of secondary and tertiary structures. Besides, variation of the rate constant with temperature gave Linear Eyring's plots. Thus, despite its irreversibility, this process resembles the kinetics of reversible protein unfolding. Due to the basicity of caricain, all of the carboxylates in the native protein interact with nearby positively charged groups. Then, it may be thought that pK values of titratable sites are mainly influenced by interactions of this type. Accordingly, we set up a simple electrostatic perturbation model, based on charge-charge interactions at distances not larger than 10 Angstrom, which reproduces reasonably well the titration curve of native caricain. Because the pH dependence of the activation free energy for unfolding (Delta G(not equal)) can be related to differences in the protonation behavior of the native (N) state with respect to the transition (TS) state, the model was further used to analyze the experimental Delta G(not equal) vs pH curve. Results from this analysis suggest that there is an increase of about 3 Angstrom in the average ion-pair distance when N globally expands to form TS. Alternatively, if the expansion were restricted to only one molecular domain, the structure of this domain in TS would be highly disordered. In either case, it is probable that the solvent-accessible area augments significantly during the expansion.