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) andL-cysteine (CySH) were trapped with 5,5′-dimethyl-1-pyrrolineN-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/•CO−2spectrum 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.