The decline of molecular activity of cytochrome oxidase during purification.

The losses of molecular activity which take place when cytochrome oxidase is purified from bovine heart muscle particles by the procedures of Yonetani ((1960) J. Biol. Chem. 235, 845) and of Fowler et al. ((1962) Biochim. Biophys. Acto 64, 170) have been examined. Bovine heart muscle particles (about 1 nmole of heme a per mg of protein) and oxidase-rich particles (about 4 nmoles of heme a per mg of protein) treated with deoxycholate under optimal conditions, had molecular activities (MAO,,BX) (at infinitely high concentration of cytochrome c, spectrophotometrically determined in 0.05 M phosphate buffer, pH 7.0, 25”) in the range of 530 to 580 s-‘. MA o,,,,~~, determined under identical reaction conditions, declined with successive steps in the Yonetani and Fowler et al. purification procedures for cytochrome oxidase, to considerably lower values for the finally isolated enzymes, while KmaPP stayed relatively unchanged. Polyacrylamide gel electrophoresis in the presence of dodecyl sulfate, showed no evidence of essential component removal during purification, that might account for decreased MA,,,,,,@, values. The accumulation of an endogenous inhibitor in the course of preparation was also shown to be an unlikely cause for the observed decline of MA,,,,,,. Treatment with limited quantities of dodecyl sulfate led to stimulation of activity of Yonetani’s preparation. Similar treatment of Fowler’s preparation and of deoxycholatetreated, oxidase-rich particles resulted only in decreased molecular activity. Dodecyl sulfate appeared to be an uncompetitive inhibitor of cytochrome oxidase. The aerobic ferrocytochrome c oxidation catalyzed by deoxycholate-solubilized particulate oxidase exhibited apparent first order kinetics over at least 99% of the reaction course. Purified oxidase preparations showed deviations from first order (concave semilogarithmic reaction plots) at low remaining substrate concentrations. This could not be ascribed either to spontaneous activation in the reaction