Fractionation and purification of hepatic microsomal cytochrome P-450 isoenzymes from phenobarbital-pretreated rats by h.p.l.c. A convenient tool for screening of isoenzymes inactivated by allylisopropylacetamide.

A procedure incorporating the salient features of ion-exchange column chromatography with ion-exchange h.p.l.c. is described for the fractionation and purification to homogeneity of several membrane-bound rat hepatic phenobarbital (PB)-inducible cytochrome P-450 isoenzymes, including the major PB-inducible species. The resolving power of this technique makes it a highly promising tool for the isolation and purification of closely related cytochrome P-450 isoenzymes. In addition, it may also be used for screening of individual isoenzymes either selectively induced or repressed by a variety of endobiotics or xenobiotics. Accordingly, we have exploited this particular feature to identify not only the PB-inducible cytochrome P-450 isoenzymes destroyed in vivo by allylisopropylacetamide, a suicide inactivator of cytochrome P-450, but also to distinguish those that are reparable by exogenous haemin from those that are irreparably damaged.