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
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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.
Patel RP et al (1999) Biological aspects of reactive nitrogen species. Biochim Biophys Acta BBA 1411:385–400CrossRefGoogle Scholar
Dedon PC, Tannenbaum SR (2004) Reactive nitrogen species in the chemical biology of inflammation. Arch Biochem Biophys 423:12–22CrossRefGoogle Scholar
Pacher P, Beckman JS, Liaudet L (2007) Nitric oxide and peroxynitrite in health and disease. Physiol Rev 87:315–424CrossRefGoogle Scholar
Ferrer-Sueta G et al (2018) Biochemistry of peroxynitrite and protein tyrosine nitration. Chem Rev 118:1338–1408CrossRefGoogle Scholar
Pfeiffer S, Schmidt K, Mayer B (2000) Dityrosine formation outcompetes tyrosine nitration at low steady-state concentrations of peroxynitrite: implications for tyrosine modification by nitric oxide/superoxide in vivo. J Biol Chem 275:6346–6352CrossRefGoogle Scholar
Pfeiffer S, Lass A, Schmidt K, Mayer B (2001) Protein tyrosine nitration in mouse peritoneal macrophages activated in vitro and in vivo: evidence against an essential role of peroxynitrite. FASEB J 15:2355–2364CrossRefGoogle Scholar
Blough NV, Zafiriou OC (1985) Reaction of superoxide with nitric oxide to form peroxonitrite in alkaline aqueous solution. Inorg Chem 24:3502–3504CrossRefGoogle Scholar
Robinson KM, Beckman JS (2005) Synthesis of peroxynitrite from nitrite and hydrogen peroxide. Methods in Enzymology. Elsevier, Amsterdam, pp 207–214Google Scholar
Pryor WA et al (1995) A practical method for preparing peroxynitrite solutions of low ionic strength and free of hydrogen peroxide. Free Radic Biol Med 18:75–83CrossRefGoogle Scholar
Bohle DS, Glassbrenner PA, Hansert B (1996) Synthesis of pure tetramethylammonium peroxynitrite. Methods in Enzymology. Elsevier, Amsterdam, pp 302–311Google Scholar
Li X et al (2015) Visualizing Peroxynitrite fluxes in endothelial cells reveals the dynamic progression of brain vascular injury. J Am Chem Soc 137:12296–12303CrossRefGoogle Scholar
Molon B et al (2011) Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells. J Exp Med 208:1949–1962CrossRefGoogle Scholar
Palmer RMJ, Ferrige AG, Moncada S (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524–526CrossRefGoogle Scholar
Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G (1987) Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci USA 84:9265–9269CrossRefGoogle Scholar
Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87:1620–1624CrossRefGoogle Scholar
Koppenol WH (2001) 100 Years of peroxynitrite chemistry and 11 years of peroxynitrite biochemistry. Redox Rep 6:339–341CrossRefGoogle Scholar
Salvemini D et al (1996) Evidence of peroxynitrite involvement in the carrageenan-induced rat paw edema. Eur J Pharmacol 303:217–220CrossRefGoogle Scholar
Salgo MG, Squadrito GL, Pryor WA (1995) Peroxynitrite causes apoptosis in rat thymocytes. Biochem Biophys Res Commun 215:1111–1118CrossRefGoogle Scholar
Saha A, Goldstein S, Cabelli D, Czapski G (1998) Determination of optimal conditions for synthesis of peroxynitrite by mixing acidified hydrogen peroxide with nitrite. Free Radic Biol Med 24:653–659CrossRefGoogle Scholar