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A practicable method to prepare nitrated proteins with peroxynitrite and low concentration of sodium hydroxide

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Abstract

Peroxynitrite is an ion acting as a powerful oxidant and nucleophile, which plays a key role in the inflammation and aging process by nitrating tyrosine or tryptophan residues of the proteins. Nitration of a target protein is considered to be a proper method to study the behavioral change of the proteins being nitrated. The commonly used methods for peroxynitrite preparation in vitro usually contain high concentration of sodium hydroxide, which easily induces hydrolysis of target proteins. Accordingly, the method for peroxynitrite preparation was optimized in vitro by changing the sequence of hydrochloric acid and sodium hydroxide added. After different amount of hydrochloric acid added to the system following sodium nitrite, peroxynitrite can be yielded in a concentration up to 60 mM with sodium hydroxide as low as 17 mM. More importantly, biological activity of the target protein was well maintained after protein nitration since low sodium hydroxide was used.

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Acknowledgements

The authors are grateful for the financial support provided by the National Program on Key Research Project of China (2016YFD0500406), the National Natural Sciences Foundation of China (Grant No. 31872455), and Fundamental Research Fund for the Central University (2662018PY016).

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Authors and Affiliations

  1. State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China

    Nan Zhang, Xiaochen Gao, Weijia Zhang, Doaa Higazy, Ke Wang, Zhenfang Fu & Min Cui

  2. Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, Hubei, China

    Nan Zhang, Xiaochen Gao, Weijia Zhang, Doaa Higazy, Ke Wang, Zhenfang Fu & Min Cui

  3. Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture of the People’s Republic of China, Wuhan, 430070, Hubei, China

    Nan Zhang, Xiaochen Gao, Weijia Zhang, Doaa Higazy, Ke Wang, Zhenfang Fu & Min Cui

  4. International Research Center for Animal Disease, Ministry of Science and Technology of the People’s Republic of China, Wuhan, 430070, Hubei, China

    Nan Zhang, Xiaochen Gao, Weijia Zhang, Doaa Higazy, Ke Wang, Zhenfang Fu & Min Cui

  5. Departments of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA, 30602, USA

    Zhenfang Fu

Authors
  1. Nan Zhang
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  2. Xiaochen Gao
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  3. Weijia Zhang
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  4. Doaa Higazy
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  5. Ke Wang
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  6. Zhenfang Fu
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  7. Min Cui
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Correspondence to Min Cui.

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Zhang, N., Gao, X., Zhang, W. et al. A practicable method to prepare nitrated proteins with peroxynitrite and low concentration of sodium hydroxide. Mol Biol Rep 47, 1393–1398 (2020). https://doi.org/10.1007/s11033-019-05211-w

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  • DOI: https://doi.org/10.1007/s11033-019-05211-w

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