Detection and Characterization of the Electron Paramagnetic Resonance-Silent Glutathionyl-5, 5-dimethyl-1-pyrroline N-oxide Adduct Derived From Redox Cycling of Phenoxyl Radicals in Model Systems and HL-60 CellsEPA Grant Number: U914824
Title: Detection and Characterization of the Electron Paramagnetic Resonance-Silent Glutathionyl-5, 5-dimethyl-1-pyrroline N-oxide Adduct Derived From Redox Cycling of Phenoxyl Radicals in Model Systems and HL-60 Cells
Investigators: Goldman, Radoslav
Institution: University of Pittsburgh
EPA Project Officer: Broadway, Virginia
Project Period: January 1, 1995 through January 1, 1996
Project Amount: $102,000
RFA: STAR Graduate Fellowships (1995) Recipients Lists
Research Category: Biology/Life Sciences , Academic Fellowships , Fellowship - Biochemistry
The objectives of this research project are to: (1) develop a novel high performance liquid chromatography (HPLC) method for the detection of an electron paramagnetic resonance (EPR)-silent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) adduct of glutathionyl radicals in model systems and in cells; and (2) synthesize a sufficient quantity of the adduct for characterization by UV spectrophotometry, ionspray mass spectrometry, and 1H nuclear magnetic resonance (NMR) spectroscopy.
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 glutathionyl S-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 are not sufficiently stable, and can be detected only under steady-state conditions. The UV absorption max of the adduct, 258 nm, was indicative of a 2-(S-alkylthiyl) pyrroline N-oxide chromophore. The molecular mass of the adduct was 418 amu. No signal for the C2 proton of the DMPO-derived portion of the adduct was evident in its 1H NMR spectrum. The results were consistent with the structure 2-(S-glutathionyl)-5,5-dimethyl-1pyrroline N-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. To our knowledge, this is 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.