Electron spin resonance spin-trapping detection of superoxide generated by neuronal nitric oxide synthase

Jeannette Vásquez-Vivar, Pavel Martásek, Neil Hogg, Hakim Karoui, Bettie Sue Siler Masters, Kirkwood A. Pritchard, B. Kalyanaraman

Research output: Contribution to journalArticle

36 Scopus citations

Abstract

NOS is a ubiquitous enzyme that has an oxygenase and reductase activity. NOS reduces electron acceptors, at the reductase domain, by a one-electron mechanism that is not inhibited by SOD. One example of this activity is the direct reduction of ferricytochrome c by nNOS. Redox cycling electron acceptors (EA in Scheme 1), such as lucigenin and NBT, are reduced by NOS to generate an intermediate radical (EA(red)). This radical can then be reoxidized to the parent compound by oxygen, and in the process generate superoxide. Consequently, both NBT and lucigenin will enhance NADPH-dependent superoxide generation in the presence of flavoprotein reductases such as NOS. The artificial generation of superoxide from lucigenin and NBT is a major pitfall in the use of these compounds as superoxide probes. We conclude that the use of ESR spin-trapping techniques, although not free of problems, is a viable technique for the detection and quantification of superoxide in systems containing nNOS.

Original languageEnglish (US)
Pages (from-to)169-177
Number of pages9
JournalMethods in enzymology
Volume301
DOIs
StatePublished - 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Vásquez-Vivar, J., Martásek, P., Hogg, N., Karoui, H., Masters, B. S. S., Pritchard, K. A., & Kalyanaraman, B. (1999). Electron spin resonance spin-trapping detection of superoxide generated by neuronal nitric oxide synthase. Methods in enzymology, 301, 169-177. https://doi.org/10.1016/S0076-6879(99)01080-0