Interpretation of DSC data on protein denaturation complicated by kinetic and irreversible effects

Thermal denaturation of Kunitz soybean trypsin inhibitor (KTI) and ribulose -1,5-biphosphate carboxylase (RBPC) from tobacco leafs was studied by the m ethod of high-sensitivity differential scanning calorimetry (HS-DSC). The d ependence of the denaturation temperature on the heating rate reveals in th e case of both proteins a non-equilibrium character of the denaturation tra nsition in applied conditions. Developed kinetic approach allows the determ ination of an equilibrium transition temperature as well as the rate consta nts of denaturation and renaturation from the complex data of HS-DSC. This method was applied to the analysis of the pH-induced change of the conforma tional stability of KTI within pH range from 2.0 to 11.0. It allowed the de termination of the pH dependencies: of the excess free energy of denaturati on, of the activation enthalpy and entropy of denaturation as well as of th e denaturation rate constant. Conclusions have been made suggesting the con tribution of side-chain hydrogen bonds in the stabilisation of the native a nd activated states of KTI. (C) 2000 Elsevier Science B.V. All rights reser ved.