Isolation and characterization of cytochrome b5 from Candida tropicalis grown on alkane

Cytochrome b5 from Candida tropicalis grown on alkane has been solubulized in three different ways (sodium cholate, trypsin, osmotic wash). After solubilization of the microsomal membrane with sodium cholate, the purification of cytochrome b5 was achieved by DEAE-cellulose chromatography, hydroxylapatite chromatography, a second DEAE-cellulose chromatography and a Sephadex G-75 gel filtration. The purified protein had an apparent molecular weight of 16 000 +/- 1 000. After solubilization by trypsin treatment or osmotic wash, the purification procedure yielded a protein with an apparent molecular weight of 12 000 +/- 1 000. Though the purified proteins presented molecular weights depending on the technique of solubilization, they exhibited identical optical properties, a great stability with respect to temperature and pH, and were all autooxidable. Redox titrations revealed differences in their midpoint potential values, which were 35 +/- 5 mV for the b5 purified after cholate solubilization, -59 +/- 5 mV for the b5 purified after trypsin treatment and -65 +/- 5 mV for the b5 purified after osmotic wash.