Crystalline Cytochrome b5

Abstract

A method for purification and crystallization of cytochrome b₅ from rabbit river microsomes was worked out. For solubilization of cytochrome b₅ from microsomes, crystalline proteinases were used. The solubilized cytochrome was purified by treatments with CM-cellulose, DEAE-cellulose and chromatography on DEAE-Sephadex. In the case of trypsin [EC 3.4.4.4] solubilization, the cytochrome was separated into two fractions on the DEAE-Sephadex column and both were crystallized from ammonium sulfate solution. Yield of cytochrome b₅ from the major fraction (the faster moving band) and from the minor fraction were about 20 and 3 mg per 1kg of the liver, respectively. The former crystals were hexagonal plates and the latter were rectangular plates. In the case of solubilization by a bacterial proteinase (Nagarse), cytochrome b₅ was also separated into two fractions on column chromatography and the shape of the crystals from the major fraction (the slower moving band) was hexagonal rod. The crystalline preparation of cytochrome b₅ of the major fraction obtained by trypsin solubilization was ultracentrifugally and electrophoretically homogeneous. Its molecular weight was estimated to be 11, 500 from iron content. The results of elemental and amino acid analyses of the preparation are also reported.