Unfolding of creatine kinase induced by acid studied by isothermal titration calorimetry and fluorescence spectroscopy

Abstract

Thermodynamics of the unfolding of rabbit muscle-type creatine kinase (MM-CK) induced by acid has been studied by isothermal titration calorimetry and fluorescence spectroscopy. The conformational transition between the native state and a partially folded intermediate of this protein occurs in the pH range 7.0–5.2, and the transition between the intermediate and the unfolded state of this protein occurs in the pH range 5.2–3.0. The protein is almost fully unfolded at pH 3.0. The intrinsic molar enthalpy changes for formation of the unfolded state of MM-CK induced by acid at 15.0, 25.0, 30.0 and 37.0 °C have been determined by isothermal titration calorimetry. A large positive molar heat capacity change of the unfolding, 36.8 kJ mol −1 K −1, at all temperatures examined indicates that hydrophobic interaction is the dominant driving force stabilizing the native structure of MM-CK. Combining the results from ‘phase diagram’ method of fluorescence, we conclude that the acid-induced unfolding of MM-CK follows a ‘three-state’ model.