Isolation and partial characterization of a new soluble b-type cytochrome (b9) from rat liver

A new soluble cytochrome, designated as cytochrome b9, was purified to apparent homogeneity from rat liver. The absorption maximum of the oxidized (the native form) cytochrome b9 at room temperature was 413 nm. The dithionite-reduced cytochrome b9 had absorption maxima at 556, 527, and 423 nm. The prosthetic group of cytochrome b9 was identified as protoheme IX. From gel filtration experiments, the molecular weight of cytochrome b9 was estimated to be 125,000. Polyacrylamide gel electrophoresis experiments in the presence of sodium dodecyl sulfate showed that the molecular weight of its subunit was 61,000. The native form of cytochrome b9 was thus a dimer. The amount of heme/mol of dimer was 3.3 mol. Cytochrome b9 was autoxidizable and did not bind CO, 2.2 mM cyanide, or 2.2 mM azide. On the basis of its molecular weight of 125,000, the millimolar extinction coefficients of dimeric cytochrome b9 at 280 and 413 nm were 384 and 380, respectively. The absorbance at 280 nm/mg cytochrome b9 was 3.1. Cytochromes b9 and H-450 (I.-C. Kim and W.C. Deal (1976) Biochemistry 15, 4925-4930) are the only b-type, soluble cytochromes which have been isolated from mammalian liver; they are not found in tissues of heart, lung, kidney, and brain. The biological function of cytochrome b9 was not determined.