Urea Induced Inactivation and Unfolding of Arginine Kinase from the Sea Cucumber Stichopus japonicus

Shu-Yuan Guo¹, Zhi Guo¹, Bao-Yu Chen¹, Qin Guo¹, Shao-Wei Ni^1,2, and Xi-Cheng Wang^1,2*

¹Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, China; fax: 8610-627-72248; E-mail: wangxic@mail.tsinghua.edu.cn

²Protein Science Laboratory of the Ministry of Education, School of Life Science and Engineering, Tsinghua University, Beijing 100084, China

^* To whom correspondence should be addressed.

Received May 26, 2003; Revision received May 31, 2003

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Urea titration was used to study the inactivation and unfolding equilibrium of arginine kinase (AK) from the sea cucumber Stichopus japonicus. Both fluorescence spectral and circular dichroism spectral data indicated that an unfolding intermediate of AK existed in the presence of 1.0 to 2.0 M urea. This was further supported by the results of size exclusion chromatography. The spectral data suggested that this unfolding intermediate shared many structural characteristics with the native form of AK including its secondary structure, tertiary structure, as well as its quaternary structure. Furthermore, according to the residual activity curve, this unfolding intermediate form still retained its catalytic function although its activity was lower than that of native AK. Taken together, the results of our study give direct evidence that an intermediate with partial activity exists in unfolding equilibrium states of AK during titration with urea.

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KEY WORDS: arginine kinase, conformational change, equilibrium intermediate, urea denaturation

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