Detection, characterization, and decay kinetics of ROS and thiyl adducts of mito-DEPMPO spin trap.
We report here the detection and characterization of spin adducts formed from the trapping of reactive oxygen species (superoxide and hydroxyl radicals) and glutathiyl and carbon-centered radicals by a newly synthesized nitrone, Mito-DEPMPO. This is a cationic nitrone spin trap with a triphenyl phosphonium cation conjugated to the DEPMPO analogue. The Mito-DEPMPO-OOH adduct, formed from the trapping of superoxide by Mito-DEPMPO, was enzymatically generated using xanthine/xanthine oxidase and neuronal nitric oxide synthase, and chemically generated by KO2 in 18-crown-6. The Mito-DEPMPO-OOH adduct exhibits an eight-line EPR spectrum with partial asymmetry arising from the alternate line-width effect. The half-life of the Mito-DEPMPO-OOH adduct is 2-2.5-times greater than that of the DEPMPO-OOH. The Mito-DEPMPO-SG adduct, formed from the trapping of glutathiyl radicals by Mito-DEPMPO, is 3-times more persistent than the analogue DEPMPO-SG adduct. In this study, we describe the EPR characterization of spin adducts formed from Mito-DEPMPO. The EPR parameters of Mito-DEPMPO adducts are distinctly different and highly characteristic. The detection of superoxide from an intact mitochondrion was feasible with Mito-DEPMPO but not with DEPMPO. We conclude that Mito-DEPMPO nitrone and its analogues are more effective than most nitrone spin traps for trapping superoxide, hydroxyl, and thiyl radicals formed in biological systems, including mitochondria.
Hardy
Micaël
M
Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Rockenbauer
Antal
A
Vásquez-Vivar
Jeannette
J
Felix
Christopher
C
Lopez
Marcos
M
Srinivasan
Satish
S
Avadhani
Narayan
N
Tordo
Paul
P
Kalyanaraman
B
B
D000818:Animals; D002460:Cell Line; D004578:Electron Spin Resonance Spectroscopy; D017665:Hydroxyl Radical / Q000032:analysis / Q000737:chemistry*; D007700:Kinetics; D051379:Mice; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D011758:Pyrroles / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000032:analysis / Q000737:chemistry*; D019097:Spin Trapping / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000737:chemistry*; D013482:Superoxide Dismutase / Q000737:chemistry / Q000378:metabolism; D013481:Superoxides / Q000032:analysis / Q000737:chemistry*
C510912:(diethoxyphosphoryl)-5-methylpyrroline N-oxide; D011758:Pyrroles; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D013481:Superoxides; D017665:Hydroxyl Radical; D013482:Superoxide Dismutase