Affinity chromatography of microsomal enzymes on immobilized detergent-solubilized cytochrome b5

Highly selective chromatography of microsomal enzymes has been carried out on columns of immobilized cytochrome b5 that was obtained by detergent solubilization (d-b5) of the complete amphipathic molecule. Several partially purified isozymes of cytochrome P-450 are resolved on d-b5 columns, and one high-affinity isozyme has been readily purified to homogeneity. Chromatographic selectivity and correlation of elution order of isozymes of cytochrome P-450 with direct spectral measurements of affinity constants suggests affinity chromatography on d-b5 columns. Substantial one-step enrichments of NADH-cytochrome-b5 reductase and an unstable cytochrome b5-dependent oxidase of cholesterol synthesis, 4-methyl sterol oxidase, have been obtained on d-b5 columns which further supports this conclusion. Comparison of chromatographic behavior on columns of immobilized cytochrome b5 that was obtained by trypsin solubilization (t-b5) with d-b5 columns shows marked differences which must be attributed to the absence of the hydrophobic domain of the t-b5 molecule. NADH-cytochrome-b5 reductase and the high affinity isozyme of cytochrome P-450 purified by d-b5 affinity chromatography are poorly retained on t-b5 columns. A different cytochrome P-450 isozyme with lower affinity for cytochrome b5 is only retained on d-b5 columns. Cytochrome-P-450 reductase is not retained on either column. Because affinity chromatography is suggested on d-b5 columns, the procedure may be generally applicable for predicting protein-protein interactions of microsomal electron transport components that either donate electrons to, or receive electrons from, cytochrome b5. In addition, the procedure should have considerable utilitarian application for enzyme enrichment.