Generation of superoxide radicals by heart mitochondria: a study by the method of spin trapping under conditions of constant oxygenation

The generation of superoxide radicals by isolated rat heart mitochondria wa s studied by the spin trapping technique. The sample was placed into the ca vity of an EPR spectrometer in a thin-wall tenon capillary tube, which made it possible to maintain the partial oxygen pressure in the mitochondrial s uspension at a constant level. Tiron was used as a spin trap, and the inten sity of its EPR signal corresponded to the rate of O-2(.-) formation in the sample. The addition of oxidation substrates (succinate, glutamate, and ma late) into the incubation mixture caused the appearance of the Tiron EPR si gnal. The rate of superoxide radical generation by heart mitochondria stron gly increased in the presence of antimycin A, an inhibitor of the Q-cycle i n complex III of the respiratory chain, but it was completely depressed by another inhibitor of Q-cycle myxothiazol, The inhibition of the reverse ele ctron transport in complex I of the respiratory chain by rotenone (oxidatio n substrate - succinate) caused a substantial decrease in the rate of O-2(. -) formation by mitochondria.