Purification of cytochromes P-450 derived from liver microsomes of untreated and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated marmoset monkeys.
The purification of multiple forms of cytochrome P-450 (P450) from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated marmosets using fast protein liquid chromatography (FPLC) is described. The main aim was to achieve a better separation of certain closely related P450 sub-forms from each other than that previously obtained using conventional chromatography. An 8-aminooctyl-Sepharose fraction of cholate-solubilized microsomes was obtained first and, after fast desalting on Sephadex G-25, loaded on to a preparative Mono Q column. Five of the six gradient peaks contained P450 and were each rechromatographed on an analytical Mono Q column. The pass-through peak was fractionated further using a Mono S column. Other HPLC-quality anion- and cation-exchange gels were compared. For removal of excess of non-ionic detergent, five types of hydroxyapatite gels were compared. Seven purified forms of P450 and cytochrome b5 and P420 were isolated and characterized according to PHAST sodium dodecylsulphate-polyacrylamide gel electrophoretic apparent molecular masses, catalytic, spectral and magnetic properties and also TCDD-binding capacity (molar ratio of [14C]TCDD to P450). There are at least two sub-forms which appear to be TCDD inducible, one showing a substantial ethoxyresorufin-O-deethylase activity and the other having a high TCDD-binding molar ratio. Two other forms appear to be constitutive, as deduced from comparisons with forms purified from untreated animals.
Institut für Toxikologie und Embryopharmakologie, Freie Universität Berlin, Germany.
M
M
Kastner
D
D
Neubert
D000818:Animals; D002144:Callithrix; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D008862:Microsomes, Liver / Q000201:enzymology*; D000072317:Polychlorinated Dibenzodioxins / Q000008:administration & dosage*
D000072317:Polychlorinated Dibenzodioxins; D003577:Cytochrome P-450 Enzyme System