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J. Biol. Chem., Vol. 278, Issue 32, 30098-30105, August 8, 2003

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Unfolding of Rabbit Muscle Creatine Kinase Induced by Acid

A STUDY USING ELECTROSPRAY IONIZATION MASS SPECTROMETRY, ISOTHERMAL TITRATION CALORIMETRY, AND FLUORESCENCE SPECTROSCOPY^*

Yi Liang  , Fen Du , Sarah Sanglier ¶, Bing-Rui Zhou , Yi Xia , Alain Van Dorsselaer ¶, Clarisse Maechling ||, Marie-Claude Kilhoffer || and Jacques Haiech ||

From the College of Life Sciences, Wuhan University, Wuhan 430072, China, the ^¶Unité Mixte de Recherche CNRS 7509, Ecole Européenne de Chimie, Polymères et Matériaux, Université Louis Pasteur de Strasbourg, 67087 Strasbourg, France, and the ^||Unité Mixte de Recherche CNRS 7034, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg, 67401 Illkirch, France

Electrospray ionization mass spectrometry, isothermal titration calorimetry (ITC), fluorescence spectroscopy, and glutaraldehyde cross-linking SDS-PAGE have been used to study the unfolding of rabbit muscle creatine kinase (MM-CK) induced by acid. The mass spectrometric experiments show that MM-CK is unfolded gradually when titrated with acid. MM-CK is a dimer (the native state) at pH 7.0 and becomes an equilibrium mixture of the dimer and a partially folded monomer (the intermediate) between pH 6.7 and 5.0. The dimeric protein becomes an equilibrium mixture of the intermediate and an unfolded monomer (the unfolded state) between pH 5.0 and 3.0 and is almost fully unfolded at pH 3.0 reached. The results from a "phase diagram" method of fluorescence show that the conformational transition between the native state and the intermediate of MM-CK occurs in the pH range of 7.0–5.2, and the transition between the intermediate and the unfolded state of the protein occurs between pH 5.2 and 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 ITC. A large positive molar heat capacity change of the unfolding, 8.78 kcal 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 these four methods, we conclude that the acid-induced unfolding of MM-CK follows a "three-state" model and that the intermediate state of the protein is a partially folded monomer.

Received for publication, April 17, 2003 , and in revised form, May 22, 2003.

^* This work was supported by the 973 Project from the Chinese Minister of Science and Technology (Grant G1999075608), by the National Natural Science Foundation of China (Grant 39970164), and by the France-China cooperation program from Université Louis Pasteur de Strasbourg. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 86-27-8721-4902; Fax: 86-27-8788-2661; E-mail: liangyi{at}whu.edu.cn.

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