Immobilization of pig liver microsomes. Stability of cytochrome P-450-dependent monooxygenase activities

Abstract

Microsomes from pig liver were covalently coupled to Sepharose activated by CNBr and to Sephadex activated by 1,1'-carbonyldiimidazole. Microsomes were also entrapped inside Ca-alginate and kappa-carrageenan gels. The concentration of immobilized cytochrome P-450 was determined by CO-difference spectra. The activity of the monooxygenase system was demonstrated by the N-demethylation of aminopyrine, the O-demethylation of p-nitroanisole, and the hydroxylation of perhexiline maleate. Upon immobilization, a 30-40% and a 60-70% decrease in Vappmax for the O- and N-demethylations were respectively observed. The Vappmax values for the hydroxylation of perhexiline maleate were essentially the same for the different immobilized forms and for the freely suspended microsomal cytochrome P-450. Under storage at 4 degrees C, microsomes entrapped inside kappa-carrageenan and Ca-alginate were less stable than the free microsomes, whereas immobilization on CNBr-activated Sepharose improved the stability of the hepatic microsomal monooxygenase system at the same temperature. These types of immobilized microsomes have the advantage of being easily recovered and reused for other assays. Finally, microsomes entrapped inside kappa-carrageenan or Ca-alginate can be used to follow up the continuous metabolization of p-nitroanisole for several hours in a stirred-batch reactor.