Effect of Zn²⁺ during Reactivation and Refolding of Urea-Denatured Creatine Kinase

S. Li¹, Z. Xu¹, and H.-M. Zhou^1,2*

¹Department of Biological Science and Biotechnology, Tsinghua University, Beijing, 100084 P. R. China; fax: (8610) 62772245; E-mail: zhm-dbs@mail.tsinghua.edu.cn

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

^* To whom correspondence should be addressed.

Received March 16, 2001

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The courses of refolding and reactivation of urea-denatured creatine kinase (CK) (ATP:creatine N-phosphotransferase, EC 2.7.3.2) have been studied in the absence and presence of zinc ions. The presence of Zn²⁺ at low concentrations blocks the reactivation and refolding of urea-denatured CK and keeps it in a partially folded state. The partially folded state proved to be a monomeric state which resembles the “molten globule” state in the CK folding pathway. During refolding in the presence of Zn²⁺, creatine kinase forms aggregates with the aggregation dependent on zinc concentration and temperature. In the presence of EDTA, the partially folded creatine kinase can be reactivated and refolded following a biphasic course, suggesting the existence of a monomeric intermediate during the refolding of CK. The results also suggest that low concentrations of zinc ions might be toxic to some proteins such as creatine kinase by disrupting their proper folding.

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KEY WORDS: creatine kinase, zinc ion, reactivation, refolding, partially folded state

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