Superoxide reaction with nitroxides.
Stable, free radical nitroxides are commonly used ESR spectroscopy tools. However, it has recently been found that ESR observable signal from 5-membered ring spin-adducts or stable label nitroxides is lost or diminished by reaction with superoxide. A similar radical-radical annihilation was not found for six membered ring nitroxide radicals. To discern why six-membered ring nitroxides are not reduced under superoxide flux generated by hypoxanthine/xanthine oxidase, spectrophoptmetric (Cyt CIII) and chemiluminescence (lucigenin) and ESR assays were used to follow the reactions. Spectrophotometry and chemiluminescence clearly demonstrated that the six-membered piperidine-1-oxyl compounds (TEMPO, TEMPOL, and TEMPAMIN) rapidly react with superoxide: rate constants at pH 7.8 ranging from 7 x 10(4) to 1.2 x 10(5) M-1 s-1. The absence of detectable ESR signal loss results from facile re-oxidation of the corresponding hydroxylamine by superoxide. To fully corroborate the efficiency of the 6-membered nitroxide superoxide dismutase activity, they were shown to protect fully mammalian cells from oxidative damage resulting from exposure to the superoxide and hydrogen peroxide generating system hypoxanthine/xanthine oxidase. Since six-membered cyclic nitroxides react with superoxide about 2 orders of magnitude faster than the corresponding 5-membered ring nitroxides, they may ultimately be more useful as superoxide oxide dismutase mimetic agents.
Samuni
A
A
Molecular Biology, Hebrew University Medical School, Jerusalem, Israel.
Krishna
C M
CM
Mitchell
J B
JB
Collins
C R
CR
Russo
A
A
D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003497:Cyclic N-Oxides / Q000138:chemical synthesis; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D009589:Nitrogen Oxides / Q000378:metabolism / Q000494:pharmacology; D010080:Oxazoles / Q000138:chemical synthesis; D015203:Reproducibility of Results; D013141:Spiro Compounds / Q000138:chemical synthesis; D013481:Superoxides / Q000037:antagonists & inhibitors / Q000378:metabolism
D003497:Cyclic N-Oxides; D005609:Free Radicals; D009589:Nitrogen Oxides; D010080:Oxazoles; D013141:Spiro Compounds; D013481:Superoxides; C065425:2-spirocyclohexane doxyl (2-spirocyclohexane-5,5-dimethyl-3-oxazolidinoxyl); C034590:2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl