[Quantitative determination of the rate of superoxide radical formation in mitochondrial membranes by electron...

The reaction of hydroxylamine (1-hydroxy-2,2,6,6-tetramethyl-4-oxopiperidine) with O2-. resulting in the stable nitroxyl radical formation recorded by ESR-technique was applied to estimate quantitatively the rate of O2-. superoxide radical generation (VO2.-) by submitochondrial particles (SMP) of liver (of mice and rats). The VO2.- dependence on concentrations of NADH, succinate and protein of SMP was established. The method allows detecting VO2.- greater than 0.05 nmol.min-1.ml-1. It has been shown that in the NADH-dependent site of the chain VO2.- is 3-4 times that in the succinate-dependent site. In the presence of rhotenone and antimycin A VO2.- increases by 30-35%, while cyanide retards VO2.- by about 30%. The data comparison with regard to VO2.- and O2 absorption rate polarographically determined has indicated that about 2% of the absorbed O2 is consumed to form O2-.