Direct observation of the enthalpy change accompanying the native to molten-globule transition of cytochrome c by using isothermal acid-titration calorimetry

The enthalpy change accompanying the reversible acid-induced transition from the native (N) to the molten-globule (MG) state of bovine cytochrome c was directly evaluated by isothermal acid-titration calorimetry (IATC), a new method for evaluating the pH dependence of protein enthalpy. The enthalpy change was 30 kJ/mol at 30 °C, pH 3.54, with 500 mM KCl. The results of the global analysis of the temperature dependence of the excess enthalpy from 20 to 35 °C demonstrated that the N to MG transition is a two-state transition with a small heat capacity change of 1.1 kJ K−1 mol−1. The present findings were also indicative of the pH dependence of the enthalpy and the heat capacity of the MG state, −13 kJ mol−1 pH−1 and −1.0 kJ K−1 mol−1 pH−1, respectively, at 30 °C within a pH range from 2 to 3.