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

doi:10.1006/abbi.1996.0219

Archives of Biochemistry and Biophysics

Volume 330, Issue 1, 1 June 1996, Pages 3-11

https://doi.org/10.1006/abbi.1996.0219 Get rights and content

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 λ_maxof 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.

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¹: Current address: Department of Biochemistry, Mount Sinai Medical Center, New York, NY 10029.

²: To whom correspondence should be addressed at Department of Environmental & Occupational Health, University of Pittsburgh, 260 Kappa Drive, Pittsburgh, PA 15238. Fax: (412) 624-1020; E-mail: [email protected].

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Regular ArticleDetection 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

Regular Article
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