Vertically integrated human P450 and oxygen sensing film for the assays of P450 metabolic activities

An assaying method of cytochrome P450 (P450 or CYP) monooxygenase activities for toxicological evaluation of drugs and environmental pollutants was developed by immobilizing P450 on an oxygen sensoring layer. Membrane fractions from E. coli expressing human P450 were entrapped in agarose or silica-based gels and immobilized on 96-well microarrays having an oxygen sensing film at the bottom. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)(3)Cl(2)). P450 activity toward the substrates was monitored through the fluorescence intensity enhancement due to the oxygen consumption by the metabolic reactions. For the metabolism of chlortoluron, a selective herbicide used to control grass weeds, CYP1A1 immobilized in agarose gel showed a higher activity and stability compared with those in silica gels and free suspensions. The luminescence changing rate evaluated by the dynamic transient method (DTM) could be correlated with the substrate concentration. We also compared the metabolic responses of human P450s (CYP1A1,CYP2C8, CYP2E1, CYP3A4) toward various substances. The use of immobilized P450 on an oxygen sensing layer provides a versatile assaying platform owing to the following features. First, the oxygen sensor can detect metabolic reactions of any P450 species, in contrast with assays using fluorogenic substrates. Second, vertical integration of the oxygen sensor and immobilized P450 enhanced the sensitivity because of the effective depletion of oxygen in the vicinity of the oxygen sensing layer. Third, immobilization enables repeated use of P450 by replacing the substrate solutions using a flow cell. Furthermore, the activity of immobilized P450 was retained at least for 3 weeks at 4 °C, suggesting its long-term stability, which is an additional attractive feature.