AN ELECTRON-PARAMAGNETIC-RESONANCE STUDY OF THE ANTIOXIDANT PROPERTIES OF THE NITROXIDE FREE-RADICAL TEMPO

Previous reports of superoxide scavenging and ferroxidase-like activit y of nitroxide free radicals have greatly increased interest in the ab ility of these compounds to protect cells against oxidative cellular d amage. In the present study we investigated the antioxidant properties of the six membered nitroxide 2,2,6,6-tetramethylpiperidine-1-oxyl (T EMPO) in various assays. TEMPO (5 mM) inhibited the hydroxyl radical m ediated 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 stron g implications for the interpretation of the ferricytochrome c reducti on assay. TEMPO may be reduced to an electron paramagnetic resonance s ilent hydroxylamine by a number of pathways. TEMPO absorption intensit y decay (TAID) was monitored to investigate the effects of hydrogen pe roxide on Cu,Zn-SOD. TEMPO was found to effectively scavenge or suppre ss formation of hydroxyl radicals inside Cu,Zn-SOD. The generation of hydroxyl radical was confirmed by employing the conventional spin trap ping agent DMPO. Using radical scavengers unable to penetrate the Cu,Z n-SOD enzyme (e.g., mannitol, ethanol, albumin) or compounds with acce ss to copper within the Cu,Zn-SOD enzyme (azide and cyanide), we could not detect hydroxyl radicals outside the enzyme. Finally, since the e lectron paramagnetic resonance absorption intensity is directly propor tional to the concentration of TEMPO spins, loss of absorption intensi ty provided information concerning radical-mediated processes. Therefo re, the decay kinetics of TEMPO may be used as a very sensitive altern ative to conventional spin traps.