Nitroxides Scavenge Myeloperoxidase-Catalyzed Thiyl Radicals in Model Systems and in Cells
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- 作者：Grigory G. Borisenko ; Ian Martin ; Qing Zhao ; Andrew A. Amoscato ; and Valerian E. Kagan
- 刊名：Journal of the American Chemical Society
- 出版年：2004
- 出版时间：August 4, 2004
- 年：2004
- 卷：126
- 期：30
- 页码：9221 - 9232
- 全文大小：325K
- 年卷期：v.126,no.30(August 4, 2004)
- ISSN：1520-5126
文摘
Nitroxide radicals possess important antioxidant activity in live tissues because of their ability toscavenge reactive radicals. Despite the fact that, in cells, damaging free radicals are primarily quenchedby glutathione (GSH) with subsequent formation of harmful glutathionyl radical (GS
), interactions of nitroxideradicals with GS and thiols have not been studied in detail. In addition, intracellular metabolic pathwaysleading to the formation of secondary amines from nitroxides are unknown. Here we report that GS radicalsreact efficiently and irreversibly with nitroxides to produce secondary amines. We developed a sensitivemethod for the detection of GS based on their specific interaction with Ac-Tempo, a nonfluorescent conjugateof fluorogenic acridine with paramagnetic nitroxide Tempo, and used it to characterize interactions betweennitroxide and thiyl radicals generated through phenoxyl radical recycling by peroxidase. During reaction ofAc-Tempo with GS, Tempo EPR signals decayed and acridine fluorescence concurrently increased. DMPOand PBN, spin traps for GS, inhibited this interaction. Using combined HPLC and mass spectrometry, wedetermined that 90% of the Ac-Tempo was converted into fluorescent acridine (Ac)-piperidine; GSH wasprimarily oxidized into sulfonic acid. In myeloperoxidase-rich HL-60 cells, Ac-piperidine fluorescence wasobserved upon stimulation of GS generation by H2O2 and phenol. Development of fluorescence wasprevented by preincubation of cells with the thiol-blocking reagent N-ethylmaleimide as well as withperoxidase inhibitiors. Furthermore, Ac-Tempo preserved intracellular GSH and protected cells from phenol/GS toxicity, suggesting a new mechanism for the free-radical scavenging activity of nitroxides in live cells.
), interactions of nitroxideradicals with GS and thiols have not been studied in detail. In addition, intracellular metabolic pathwaysleading to the formation of secondary amines from nitroxides are unknown. Here we report that GS radicalsreact efficiently and irreversibly with nitroxides to produce secondary amines. We developed a sensitivemethod for the detection of GS based on their specific interaction with Ac-Tempo, a nonfluorescent conjugateof fluorogenic acridine with paramagnetic nitroxide Tempo, and used it to characterize interactions betweennitroxide and thiyl radicals generated through phenoxyl radical recycling by peroxidase. During reaction ofAc-Tempo with GS, Tempo EPR signals decayed and acridine fluorescence concurrently increased. DMPOand PBN, spin traps for GS, inhibited this interaction. Using combined HPLC and mass spectrometry, wedetermined that 90% of the Ac-Tempo was converted into fluorescent acridine (Ac)-piperidine; GSH wasprimarily oxidized into sulfonic acid. In myeloperoxidase-rich HL-60 cells, Ac-piperidine fluorescence wasobserved upon stimulation of GS generation by H2O2 and phenol. Development of fluorescence wasprevented by preincubation of cells with the thiol-blocking reagent N-ethylmaleimide as well as withperoxidase inhibitiors. Furthermore, Ac-Tempo preserved intracellular GSH and protected cells from phenol/GS toxicity, suggesting a new mechanism for the free-radical scavenging activity of nitroxides in live cells.