Detection of thiyl radical adducts formed during hydroxyl radical- and peroxynitrite-mediated oxidation of thiols - A high resolution ESR spin- trapping study at Q-band (35 Ghz)

B. Kalyanaraman, Hakim Karoui, Ravinder Jit Singh, Christopher C. Felix

Research output: Contribution to journalArticle

46 Scopus citations

Abstract

Thiyl radicals (RS(·)) formed during peroxynitrite- or hydroxyl radical-dependent oxidation of thiols, i.e., glutathione (GSH) and L-cysteine (CySH) were trapped with 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and analyzed by X-band and Q-band electron spin resonance (ESR) spectroscopy. At X-band, the ESR parameters of DMPO-glutathionyl radical adduct (DMPO/(·)SG) and DMPO-hydroxyl radical adduct (DMPO/(·)OH) are nearly similar in aqueous solutions and as a result, except for the low-field spectral line, the remaining spectral lines of DMPO/(·)SG virtually over-lap with those of the DMPO/(·)OH adduct. In contrast, at Q-band, most of the spectral lines due to the DMPO/(·)SG were separated from the DMPO/(·)OH. Inclusion of a superoxide dismutase (SOD) mimic completely abolished the formation of the DMPO/(·)OH adduct and not the DMPO/(·)SG adduct during ONOO mediated oxidation of GSH and DMPO. In the presence of formate, the DMPO/(·)SG spectrum was replaced by the DMPO/(·)CO2 spectrum which was monitored by Q- band ESR spectroscopy. Thus, spin-trapping at Q-band provides unambiguous proof for the glutathionyl radical-dependent oxidation of formate by peroxynitrite. High resolution Q-band ESR spectra of DMPO/(·)Scys were also obtained. Biological applications of the Q-band spin-trapping technique to detect thiyl radicals in cellular systems are discussed.

Original languageEnglish (US)
Pages (from-to)75-81
Number of pages7
JournalAnalytical Biochemistry
Volume241
Issue number1
DOIs
StatePublished - Oct 1 1996

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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