Detection and Characterization of the Electron Paramagnetic Resonance-Silent Glutathionyl-5,5-dimethyl-1-pyrrolineN-Oxide Adduct Derived from Redox Cycling of Phenoxyl Radicals in Model Systems and HL-60 Cells

Abstract

The antioxidant function of glutathione includes enzymatic reduction of hydrogen peroxide by glutathione peroxidase and nonenzymatic reduction of organic radicals and reactive oxygen species. The glutathionylS-centered radical, formed by the nonenzymatic reduction process, is a marker of oxidative reactions proceeding by radical mechanisms. Spin-adducts of glutathionyl radicals with the spin trap DMPO, 5,5-dimethyl-1-pyrrolineN-oxide, are not sufficiently stable and can be detected only under steady-state conditions. We developed a novel HPLC method for the detection of an EPR-silent DMPO adduct of glutathionyl radicals in model systems and in cells. We synthesized a sufficient quantity of the adduct for characterization by UV spectrophotometry, ionspray mass spectrometry, and1H NMR spectroscopy. The UV absorption λmaxof the adduct, 258 nm, was indicative of a 2-(S-alkylthiyl)pyrrolineN-oxide chromophore. The molecular mass of the adduct was 418 amu. No signal for the C2proton of the DMPO-derived portion of the adduct was evident in its1H NMR spectrum. The results were consistent with the structure 2-(S-glutathionyl)-5,5-dimethyl-1-pyrrolineN-oxide (GS-DMPO nitrone). We showed that this adduct accumulated in the course of peroxidase-dependent redox cycling of phenol in the presence of glutathione and DMPO as well as in HL-60 cells exposed to a phenol/H2O2/DMPO reaction mixture. The EPR-silent GS-DMPO nitrone was readily assayed by HPLC under conditions incompatible with the detection of the GS-DMPO nitroxide by EPR. This is to our knowledge the first direct experimental evidence for the redox cycling of phenol in this bone marrow-derived cell line. The method may prove useful in the study of radical-driven oxidations of glutathione in various pathophysiological processes associated with radical mechanisms.