Studies on Cytochrome Oxidase

Abstract

Abstract Soluble cytochrome oxidase has been prepared from the Keilin-Hartree preparation of bovine heart by sequential fragmentation of the respiratory chain. The procedure involves a prior extraction with 1% cholate which selectively solubilizes the segment containing the antimycin A-sensitive cytochrome b-c1 segment and succinate dehydrogenase. The cytochrome oxidase remaining in the particle is solubilized by 2% cholate in the presence of 0.25 saturation of ammonium sulfate and then further purified by ammonium sulfate fractionation. The procedure can be completed in 24 hours. The first order velocity constant of the purified oxidase is approximately 16 s-1 mg-1 of protein in a 3-ml system using reduced cytochrome c as the reductant at pH 5.7. The purified preparation contains approximately 11 nmoles of heme a per mg of protein and about 20% of lipid (w/w). The ratio of copper to heme a is 1.05. Amino acid composition of two preparations of oxidase indicates the protein to be slightly basic. The oxidase exhibits essentially only one sedimenting peak in ultracentrifugation experiments; its hydrodynamic data obtained in 0.1 m phosphate, containing 0.25% Emasol-1130 and 0.25% cholate, give a molecular weight of 4.3 x 105 (lipid included). However, in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis at least five major bands were observed. Spectra of the oxidized and the reduced oxidase and the effect of Emasol-1130 on the oxidized spectrum are presented. The oxidase preparation showed absorption maxima at 830, 598, 520, 419, and 280 nm in the oxidized form and 604, 520, 444, and 280 nm in the reduced form. The position of the Soret maximum, which varies from 418 to 425 nm in the oxidized enzyme as reported in the literature, was probably due to the effect of solvent used. The oxidase with the Soret peak at the longer wave length can be reversed to 418 nm by addition of Tween-80 or decrease of the Emasol concentration.