title: 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).
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 lowfield 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.
authors.affiliation: Biophysics Research Institute, Medical College of Wisconsin, Milwaukee 53226, USA.
authors.forename: B
authors.initials: B
authors.lastname: Kalyanaraman
authors.forename: H
authors.initials: H
authors.lastname: Karoui
authors.forename: R J
authors.initials: RJ
authors.lastname: Singh
authors.forename: C C
authors.initials: CC
authors.lastname: Felix
mesh_terms: D001681:Biological Assay; D005609:Free Radicals / Q000032:analysis; D009566:Nitrates / Q000378:metabolism*; D010084:Oxidation-Reduction; D019097:Spin Trapping; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D005609:Free Radicals; D009566:Nitrates; D013438:Sulfhydryl Compounds; C039642:peroxynitric acid