Research Grants/Fellowships/SBIR

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

EPA 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 - Main Campus
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

Objective:

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.

Approach:

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 lambdamax 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.

Supplemental Keywords:

fellowship, phenoxyl radical, glutathione, glutathionyl radical, 5,5-dimethyl-1-pyrroline N-oxide, DMPO, spin trapping, electron paramagnetic resonance, EPR, redox cycling, liquid chromagraphy, mass spectrometry, LC-MS, nuclear magnetic resonance, NMR, HL-60 cells, nitrone., Scientific Discipline, Water, Chemical Engineering, Biochemistry, Engineering, Chemistry, & Physics, Environmental Engineering, nuclear magnetic resonance, bioengineering, high performance liquid chromatography