A PARTIAL SEPARATION AND CHARACTERIZATION OF CYTOCHROME OXIDASE AND CYTOCHROME b*

Numerous attempts have been made to separate the insoluble cytochromes of mammalian heart muscle. Von Euler and Hellstriim in 1939 (1) used sodium cholate and ammonium sulfate and obtained a preparation containing only cytochrome b. Straub (2) obtained a fraction with sodium cholate and ammonium sulfate in which he demonstrated cytochrome oxidase (called cytochrome a3 by him after Keilin and Hartree (3)) and very little cytochrome b. In the same year Yakushiji and Okunuki (4), also using sodium cholate and ammonium sulfate, claimed the preparation of cytochrome oxidase (called cytochrome a by them). To us the preferential solubility in sodium desoxycholate of proteins that are inactive when tested with the hydroquinone-cytochrome c system (5, 6) suggested that the various components of the insoluble complex might be separated by successive additions of small amounts of desoxycholate (7). The characterization of cytochrome oxidase and cytochrome b has rested principally on a knowledge of their absorption spectra. Keilin and Hartree (3) found bands at 600 rnp and 448 mp for reduced cytochrome a3 (which they suggest may be oxidase), at 605 mp and 452 rnp for reduced cytochrome a (the existence of which we do not consider proved), and at 564, 530, and 432 rnp for reduced cytochrome b. Keilin and Hartree (3) and Graubard (8) have suggested that cytochrome oxidase may be a copper-containing enzyme. This report deals with the details of a partial separation and purification of the cytochromes contained in an insoluble preparation of mammalian heart muscle. Cytochrome b and cytochrome oxidase are characterized with respect to their absorption spectra in both the oxidized and reduced