Isolation of cytochrome P-450 components from marmoset liver microsomes by high-performance liquid chromatography

Abstract

A fast protein liquid chromatographic (FPLC) system with pre-packed and laboratory-packed columns was used for the analytical and preparative isolation of marmoset monkey cytochrome P-450 (P450) and NADPH-P450-reductase. Chromatographic separations also allowed the recovery of cytochrome b 5, NADH- b 5-reductase and epoxide hydratase. Cholate-solubilized liver microsomes from phenobarbital-induced marmosets were crudely purified on 8-aminooctyl-Sepharose or 6-aminohexyl-Sepharose and then fractionated into several isoenzyme groups using hydroxyapatite. Further purification on Mono S or CM-Sepharose and finally on phenyl-Superose, phenyl-Sepharose or octyl-Sepharose yielded a P450 fraction which was apparently homogeneous as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the automated Phast system using silver staining. Removal of excess of non-ionic detergent was effected by hydroxyapatite columns, and this was compared with other methods. For the isolation of P450 isoenzymes from untreated marmosets, Mono Q columns were employed and yielded at least two highly purified forms. NADPH-P450-reductase was recovered from the 8-aminooctyl-Sepharose column or crudely fractionated on DEAE-Sepharose Fast Flow. Subsequent purification via 2′,5′-ADP-Sepharose and Superose 12 chromatography resulted in a homogeneous preparation.