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

  • Nan Zhang
  • Xiaochen Gao
  • Weijia Zhang
  • Doaa Higazy
  • Ke Wang
  • Zhenfang Fu
  • Min CuiEmail author
Methods Paper


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.


Method Syntheses Peroxynitrite Protein nitration CCL2 Free radical 



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).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.


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© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Agricultural Microbiology, College of Veterinary MedicineHuazhong Agricultural UniversityWuhanChina
  2. 2.Key Laboratory of Preventive Veterinary Medicine in Hubei ProvinceThe Cooperative Innovation Center for Sustainable Pig ProductionWuhanChina
  3. 3.Key Laboratory of Development of Veterinary Diagnostic ProductsMinistry of Agriculture of the People’s Republic of ChinaWuhanChina
  4. 4.International Research Center for Animal DiseaseMinistry of Science and Technology of the People’s Republic of ChinaWuhanChina
  5. 5.Departments of Pathology, College of Veterinary MedicineUniversity of GeorgiaAthensUSA

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