Reduction of the Spin-label TEMPONE by Ubiquinol in the Electron Transport Chain of Intact Rabbit Spermatozoa1

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Abstract
When the hydrophilic spin label TEMPONE (deuterated 2,2,6,6-tetramethylpiperidine-N-oxyl) was incubated with intact rabbit spermatozoa at concentrations > 0.3 .times. 109 cells/ml, the ESR signal height decreased with time. This loss of signal amplitude was reversed by the oxidizing reagent potassium dichromate, indicating that the signal loss was due to a reduction of the paramagnetic nitroxide species to the nonparamagnetic hydroxylamine. Using inhibitors that act on the respiratory chain, it was observed that, relative to controls, the rate of TEMPONE reduction was decreased in the presence of rotenone, but increased in the presence of antimycin A and potassium cyanide (KCN). Parallel studies measuring oxygen consumption showed decreases with all three inhibitors. These observations to mean that TEMPONE is reduced by ubiquinol in the respiratory chain. Supporting this conclusion is the observation that the midpoint potential of TEMPONE was determined by be +48 mV, which is close to the midpoint potential of +40 mV for the ubiquinone/ubiquinol couple. In a cell-free test system, ubiquinol reduced TEMPONE, but ubiquinone, NADH, and succinate did not.