Electron paramagnetic resonance spectroscopy with N-methyl-D-glucamine dithiocarbamate iron complexes distinguishes nitric oxide and nitroxyl anion in a redox-dependent manner: Applications in identifying nitrogen monoxide products from nitric oxide synthase

Yong Xia, A. J. Cardounel, Anatoly F. Vanin, Jay L. Zweier

Research output: Contribution to journalArticle

Abstract

Though a large number of studies indicate that nitric oxide synthase (NOS) is responsible for NO. production in biological systems, controversy still remains concerning whether NOS directly produces NO.. Schmidt et al. (PNAS 93:144492, 1996) proposed that NOS first synthesizes nitroxyl anion (NO-), which is then converted to NO. by superoxide dismutase (SOD). With electron paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron (Fe-MGD), we directly detected NO. from purified NOS in the absence of SOD (Xia et al., PNAS 94:12705, 1997). We also showed that the requirement for SOD in the previous NO. measurements appeared to be due to the high levels of exogenous superoxide production in their reaction system because of the presence of free FAD. However, it was recently questioned whether Fe-MGD can discriminate NO. from NO- (Komarov et al., FRBM 28:739-742, 2000). In this study we examined the trapping specificity of different redox forms of Fe-MGD. With Fe2+-MGD, NO. generated characteristic triplet NO.-Fe2+-MGD signals (g = 2.04, a(N) = 12.7 G), whereas NO- from Angeli's salt was EPR silent. Both NO. and NO- gave rise to NO.-Fe2+-MGD signals when Fe3+-MGD was used. Strong NO. signals were measured from purified nNOS using the NO. selective Fe2+-MGD and this was not affected by SOD. Thus, spin trapping with Fe-MGD can distinguish NO. and NO- and this depends on the redox status of the iron. The detection of NO. from purified NOS by Fe2+-MGD unambiguously reconfirms our previous report that NOS directly synthesizes NO. but not NO-. Copyright (C) 2000 Elsevier Science Inc.

Original languageEnglish (US)
Pages (from-to)793-797
Number of pages5
JournalFree Radical Biology and Medicine
Volume29
Issue number8
DOIs
StatePublished - Oct 15 2000

Keywords

  • Angeli's salt
  • Electron paramagnetic resonance
  • Free radicals
  • N-methyl-D-glucamine dithiocarbamate
  • Nitric oxide
  • Nitric oxide synthase
  • Nitroxyl
  • S-nitroso-N-acetylpenicillamine
  • Spin trapping

ASJC Scopus subject areas

  • Medicine(all)
  • Toxicology
  • Clinical Biochemistry

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