Purification of cytochrome-c oxidase retaining its pulsed form.

A new purification procedure for cytochrome-c oxidase from bovine heart mitochondria is described. The enzyme was purified by selective solubilization in Triton X-100 and subsequent hydroxyapatite and gel chromatography. The preparation was highly pure and active. The subunit composition and steady-state kinetics were found to be the same as those reported for other preparations. In contrast to most of the previously published protocols the method presented here resulted in a preparation which had a rapid intramolecular electron transfer from cytochrome a to cytochrome a3, i.e. it was found to have retained its pulsed state. This correlated with monoexponential cyanide-binding kinetics. The formation of resting kinetics and biphasic cyanide-binding kinetics was shown to be induced by a short incubation at pH 5.0.