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Effect of Cysteine Modification on Creatine Kinase Aggregation

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Abstract

We studied the effect of cysteine modification on creatine kinase (CK) aggregation as well as the kinetics of the process. We found that CK aggregation was modulated by different pH conditions in the presence of Zn2+, which is a CK aggregation trigger. The CK aggregation followed first-order kinetics, and this was effectively suppressed in acidic conditions. Even under the acidic condition, cysteine modification at the active site with using 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) induced conspicuous aggregation in a dose-dependent manner. This aggregation process is directly related with decreasing the change of transition free-energy (ΔΔG AG ). When dithiothreitol (DTT) was applied to the reaction system, the aggregates were significantly reduced: DTT treatment can fully reactivate (higher than 80%) the inactive CK that was separated from CK aggregates, whereas CK was completely inactivated by Zn2+ and DTNB. Some added osmolytes such as glycine and proline were able to successfully block CK aggregation by increasing the ΔΔG AG as well as by suppressing the hydrophobic CK surface. Our study suggests the effect of cysteine modification on the unfavorable aggregation of CK and on the aggregation process that followed first-order kinetics with the accompanying changes of transitional free energy and disruptions of the hydrophobic surface. We also demonstrate the successful protocol to block the aggregation.

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Abbreviations

CK:

muscle type creatine kinase

DTNB:

5,5′-dithiobis-2-nitrobenzoic acid

DTT:

dithiothreitol

MES:

2-[N-morpholino]ethanesulfonic acid

ANS:

1-anilinonaphthalene-8-sulfonate

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Acknowledgments

This work was supported by funds from the Natural Science Foundation of China (Grant 30221003), from the Key Technologies R & D Program of Zhejiang Province (No. 2005C22053) and from the Science and Technology Bureau of Jiaxing, Zhejiang. Dr. Fei Zou was supported by a grant from the National Basic Research Program of China (No. 2006CB504100).

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Authors and Affiliations

  1. Department of Biological Science and Biotechnology, Tsinghua University, Beijing, 100084, People’s Republic of China

    He-Chang Zou

  2. Department of Environmental Health, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, 510515, People’s Republic of China

    Zhi-Rong Lü, Fei Zou & Yong-Doo Park

  3. School of Life Science, Lanzhou University, Lanzhou, 730000, People’s Republic of China

    Ye-Jing Wang & Ying-Mei Zhang

  4. Yangtze Delta Region Institute of Tsinghua University, Jiaxing, 314050, People’s Republic of China

    Yong-Doo Park

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  1. He-Chang Zou
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Correspondence to Yong-Doo Park.

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Zou, HC., Lü, ZR., Wang, YJ. et al. Effect of Cysteine Modification on Creatine Kinase Aggregation. Appl Biochem Biotechnol 152, 15–28 (2009). https://doi.org/10.1007/s12010-008-8282-4

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  • DOI: https://doi.org/10.1007/s12010-008-8282-4

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