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.
Brandt
U
U
Institut für Physikalische Biochemie der Universität München, Federal Republic of Germany.
Schägger
H
H
von Jagow
G
G
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003486:Cyanides; D003576:Electron Transport Complex IV / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010084:Oxidation-Reduction; D012995:Solubility
D003486:Cyanides; D003576:Electron Transport Complex IV