Cytochrome b5-like hemoprotein of outer mitochondrial membrane; OM cytochrome b. I. Purification of OM cytochrome b from rat liver mitochondria and comparison of its molecular properties with those of cytochrome b5.

A cytochrome b5-like hemoprotein associated with the outer membrane of rat liver mitochondria, OM cytochrome b, was purified to homogeneity after solubilization with trypsin. OM cytochrome b was separated from microsomal cytochrome b5 by hydroxylapatite chromatography, though they were indistinguishable in DEAE-cellulose chromatography and in Sephadex G-75 gel filtration. The absorption spectra of reduced OM cytochrome b and cytochrome b5 in the visible region at liquid nitrogen temperature showed small but significant differences between them. A clear difference was also detected in amino acid composition, particularly in the contents of basic amino acids and methionine. Using rabbit antibodies prepared against purified OM cytochrome b and cytochrome b5, immunochemical comparison was also carried out. No immunological cross reaction was observed by Ouchterlony double diffusion in agar gel or in a quantitative immunoprecipitation test. It is thus concluded that OM cytochrome b is distinct from microsomal cytochrome b5.