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.
Arch Biochem Biophys
; 330(1): 3-11, 1996 Jun 01.
Article
em En
| MEDLINE
| ID: mdl-8651701
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, 5,5-dimethyl-1-pyrroline N-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, and 1H NMR spectroscopy. The UV absorption lambda 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-1-pyrroline 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. 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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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fenóis
/
Espécies Reativas de Oxigênio
/
Peroxidase
/
Óxidos N-Cíclicos
/
Glutationa
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Arch Biochem Biophys
Ano de publicação:
1996
Tipo de documento:
Article
País de afiliação:
Estados Unidos
País de publicação:
Estados Unidos