Analytical fractionation of microsomal cytochrome P-450 isoenzymes from rat liver by high-performance ion-exchange chromatography

• Published in: Journal of chromatography Vol. 521; no. 2; p. 251
• Main Author: Roos, P H
• Format: Journal Article
• Language: English
• Published: Netherlands 23-11-1990
• Subjects: Animals; Chromatography, Affinity; Chromatography, High Pressure Liquid - methods; Chromatography, Ion Exchange; Cytochrome P-450 Enzyme System - isolation & purification; Detergents; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Isoenzymes - isolation & purification; Metals; Microsomes, Liver - drug effects; Microsomes, Liver - enzymology; Phenobarbital - pharmacology; Rats; Rats, Inbred Strains
• DOI: 10.1016/0021-9673(90)85050-6

Ion-exchange Fast Protein Liquid Chromatography (FPLC) on Mono Q and Mono S was optimized for the analytical separation of microsomal cytochrome P-450 species from rat liver. The effects of detergent, pH, gradient profile and column load on resolution are demonstrated. Successive application of anion- and cation-exchange chromatography leads to eleven separated P-450 fractions. The altered microsomal P450 pattern after treatment of rats with various inducers is reflected by distinct elution profiles. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and enzymatic analysis imply that several FPLC fractions contain more than one P-450 species. Preliminary results are presented showing the suitability of immobilized metal affinity chromatography (MAC) for general P-450 fractionation and thus for the further resolution of Mono Q and Mono S fractions. Scale-up for preparative P-450 fractionation is easily done by adapting the optimized analytical FPLC procedures to Q- and S-Sepharose Fast Flow.