EPR spin-trapping and nano LC MS/MS techniques for DEPMPO/OOH and immunospin-trapping with anti-DMPO antibody in mitochondrial electron transfer system.
Mitochondrial superoxide (O(2) (-)) production is an important mediator of oxidative cellular injury and pathogenesis of many diseases such as myocardial ischemia/reperfusion. The O(2) (-) generated in mitochondria acts as a redox signal triggering cellular events including apoptosis, proliferation, and senescence. The molecular mechanism of O(2) (-) produced by electron transport chain components isolated from the inner membrane is investigated by the technique of EPR spin trapping with 5-diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), indicating that FMN/FMN-binding domain (complex I), ubiquinone (complex I and III), FAD/FAD-binding domain (complex II), and cytochrome b (complex III) control the mediation of O(2) (-) production in mitochondria. O(2) (-) generation by ETC also induces oxidative damage with protein radical formation. Immunospin-trapping with anti-DMPO antibody and subsequent mass spectrometry are used to define the specific site of oxidative damage, indicating cysteine-206 and tyrosine-177 of complex I/51 kDa FMN-binding subunit and cysteine-655 of complex II/70 kDa FAD-binding subunit are involved in specific protein radical formation caused by O(2) (-) attack.
Chen
Yeong-Renn
YR
Davis Heart and Lung Research Institute, Department of Molecular and Cellular Biochemistry, College of Medicine, Ohio State University, Columbus, Ohio, USA.
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D001665:Binding Sites; D002417:Cattle; D002853:Chromatography, Liquid; D003497:Cyclic N-Oxides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D042963:Electron Transport Complex II / Q000378:metabolism; D005609:Free Radicals / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009245:NADH Dehydrogenase / Q000378:metabolism; D036103:Nanotechnology / Q000379:methods*; D018384:Oxidative Stress; D010455:Peptides / Q000737:chemistry; D011758:Pyrroles / Q000737:chemistry*; D019097:Spin Trapping / Q000379:methods*; D013481:Superoxides / Q000378:metabolism
C510912:(diethoxyphosphoryl)-5-methylpyrroline N-oxide; D000906:Antibodies; D003497:Cyclic N-Oxides; D005609:Free Radicals; D010455:Peptides; D011758:Pyrroles; D013481:Superoxides; D042963:Electron Transport Complex II; D009245:NADH Dehydrogenase