An electron paramagnetic resonance study of the antioxidant properties of the nitroxide free radical TEMPO

Previous reports of superoxide scavenging and ferroxidase-like activity of nitroxide free radicals have greatly increased interest in the ability of these compounds to protect cells against oxidative cellular damage. In the present study we investigated the antioxidant properties of the six membered nitroxide 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) in various assays. TEMPO (5 mM) inhibited the hydroxyl radical mediated generation of ethylene from 2-keto-4-methylthiobutyric acid to 31.2 +/- 4.0% of control values. Furthermore, we noted that TEMPO had the ability to maintain iron in its ferric form, a finding with strong implications for the interpretation of the ferricytochrome c reduction assay. TEMPO may be reduced to an electron paramagnetic resonance silent hydroxylamine by a number of pathways. TEMPO absorption intensity decay (TAID) was monitored to investigate the effects of hydrogen peroxide on Cu,Zn-SOD. TEMPO was found to effectively scavenge or suppress formation of hydroxyl radicals inside Cu,Zn-SOD. The generation of hydroxyl radical was confirmed by employing the conventional spin trapping agent DMPO. Using radical scavengers unable to penetrate the Cu,Zn-SOD enzyme (e.g., mannitol, ethanol, albumin) or compounds with access to copper within the Cu,Zn-SOD enzyme (azide and cyanide), we could not detect hydroxyl radicals outside the enzyme. Finally, since the electron paramagnetic resonance absorption intensity is directly proportional to the concentration of TEMPO spins, loss of absorption intensity provided information concerning radical-mediated processes. Therefore, the decay kinetics of TEMPO may be used as a very sensitive alternative to conventional spin traps.