Molecular and Cellular Biochemistry

, Volume 233, Issue 1–2, pp 57–63 | Cite as

Nitration/S-nitrosation of proteins by peroxynitrite-treatment and subsequent modification by glutathione S-transferase and glutathione peroxidase

  • Wu-Nan Kuo
  • Joseph M. Kocis


In various peroxynitrite (PN)-treated proteins, the formations of stable 3-nitrotyrosine (nitration) and labile S-nitrosocysteine (S-nitrosation) were observed by employing rapid Western blot in 6 h. The steps of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and membrane-blotting were performed at 4°C. It was noted that the intensity of immunoreactive bands specific for anti-nitrotyrosine was stronger than that specific for anti-S-nitrosocysteine. Additionally, the intensity was in the manner of a dose-dependency of PN. Nitration/S-nitrosation were formed in the following treated proteins, including bovine serum albumin (BSA), DNase-1, ceruloplasmin, catalase and hemoglobin (Hb). The incubation of PN-pretreated hemoglobin with 1 mM reduced glutathione (GSH) did not change immunoreactivity significantly. However, the addition of glutathione S-transferase (GST) or glutathione peroxidase (GPX) to the above incubation mixture, resulted in decreased immunoreactivity, suggesting GSH may form a transition complex with PN-pretreated hemoglobin and/or partially reduce/modify the treated hemoglobin, thereby increasing the accessibility for the subsequent modification by GST or GPX. Such decreased immunoreactivity indicates that nitrotyrosine and S-nitrosocysteine of treated hemoglobin was, indeed, further modified via (a) converting –NO2 to –NH2 in tyrosine residues, (b) denitrating –NO2 directly/indirectly in tyrosine residues, and/or (c) changing –S-NO to –SH in cysteine residues, or denitrosation. The findings imply similar enzymatic modifications of proteins may also occur in vivo, and therefore play a pivotal role in the NO-related cellular signaling cascade(s).

protein nitration/denitration protein S-nitrosation/denitrosation gluthatione S-transferase glutathione peroxidase 


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© Kluwer Academic Publishers 2002

Authors and Affiliations

  • Wu-Nan Kuo
  • Joseph M. Kocis

There are no affiliations available

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