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Protein Science (2004), 13:1892-1901. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Unassisted refolding of urea-denatured arginine kinase from shrimp Feneropenaeus chinensis: Evidence for two equilibrium intermediates in the refolding pathway

Ji-Cheng Pan1, Zhenhang Yu1, Xiao-Yang Su1, Ye-Qing Sun4, Xue-Ming Rao3 and Hai-Meng Zhou1,2

1 Department of Biological Science and Biotechnology and
2 Protein Science Laboratory of the Ministry of Education, School of Life Science and Engineering, Tsinghua University, Beijing 100084, China
3 College of Agronomy, Henan Agricultural University, Zhengzhou, Henan 450002, China
4 Department of Engineering and Life Science, Harbin Institute of Technology, Harbin 100051, Heilongjiang, China

(RECEIVED September 28, 2003; FINAL REVISION March 24, 2004; ACCEPTED March 25, 2004)



Abstract

The refolding process and the equilibrium intermediates of urea-denatured arginine kinase (AK) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) intrinsic fluorescence, far-UV circular dichroism (CD), size-exclusion chromatography (SEC), and enzymatic activity. In dilute denaturant, two equilibrium refolding intermediates (I and N') were discovered, and a refolding scheme of urea-denatured AK was proposed. During the refolding of urea-denatured AK, the fluorescence intensity increased remarkably, accompanied by a significant blue shift of the emission maximum and a pronounced increase in molar ellipticity of CD at 222 nm. The first folding intermediate (I) was inactive in urea solution ranging between 2.4 and 3.0 M. The second (N') existed between a 0.4- and 0.8-M urea solution, with slightly increased activity. Neither the blue shift emission maximum nor the molar ellipticity of CD at 222 nm showed significant changes in these two regions. The two intermediates were characterized by monitoring the ANS binding ability in various residual urea solutions, and two peaks of the emission intensity were observed in urea solutions of 0.6 and 2.8 M, respectively. The SEC results indicated that a distribution coefficient (KD) platform existed in urea solutions ranging between 2.4 and 3.0 M urea, suggesting that there was a similarly apparent protein profile and size in the urea solution region. The refolding kinetics showed that the urea-denatured AK was in two-phase refolding. Proline isomerization occurred in the unfolding process of AK, which blocked the slow phase of refolding. These results suggested that the refolding process of urea-denatured AK contained at the least two equilibrium refolding intermediates.

Keywords: arginine kinase; urea-denatured; refolding; equilibrium intermediate

Abbreviations: AK, arginine kinase • ANS, 1-anilino-8-naphthalenesulfonate • CD, circular dichroism • TCA, trichloroacetic acid • DTT, dithiothreitol • PAGE, sulfate-polyacrylamide gel electrophoresis • SDS, sodium dodecyl sulfate • KD, distribution coefficient • PKs, phosphagen kinases • CK, creatine kinase • GK, glycocyamine kinase • LK, lombricine kinase • UV, ultraviolet.

Reprint requests to: Hai-Meng Zhou; e-mail: zhm-dbs{at}mail.tsinghua.edu.cn; fax: 86-10-627-7-2245.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03464804.


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