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 37.0degreesC 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. (C) 2003 Elsevier B.V. All rights reserved.