89] Cytochrome b5

Abstract

Publisher Summary This chapter discusses the assay method, purification, and properties of Cytochrome b5. The procedure is described under NADH-cytochrome b5 reductase. Preparations from calf liver appears to be homogeneous by sedimentation and diffusion, but on starch gel or cellulose acetate electrophoresis, there are two major components of identical spectral and enzymatic properties that differ only in a dipeptide sequence at the carboxyl terminal end. They can be separated by careful column chromatography, but this is not necessary for normal enzymatic applications. The simpler procedure is therefore described here in which all steps are carried out at 0-5°. The steps of purification procedure described are: isolation of microsomes; incubation with pancreatic lipase; ammonium sulfate fractionation; reprecipitation at low pH; and column fractionation. The calf liver cytochrome b5 preparations appear to be homogeneous by sedimentation and in view of the fact that the protein contains one mole each of heme and tryptophan. Cytochrome b5, from both rabbit and calf liver, is stable at 20-37° between pH 7.0 and 9.5, and can be stored indefinitely at –12°. Cytochrome b5 is reduced by potassium borohydride, reduced indigo sulfonates, benzyl viologen, sodium hydrosulfite, and cysteine. Cytochrome b5 isolated from the rat, rabbit, calf, and pig liver can been detected in preparations from kidney, pancreas, adrenal medulla, mammary gland, ovary, and intestinal mucosa.