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