1 research outputs found
Microarray of Human P450 with an Integrated Oxygen Sensing Film for High-Throughput Detection of Metabolic Activities
A microarray chip containing human P450 isoforms was
constructed
for the parallel assay of their metabolic activities. The chip had
microwells that contained vertically integrated P450 and oxygen sensing
layers. The oxygen sensing film was made of an organically modified
silica film (ORMOSIL) doped with trisÂ(4,7-diphenyl-1,10-phenanthroline)
ruthenium dichloride (RuÂ(dpp)<sub>3</sub>Cl<sub>2</sub>). Human P450s
(23 types) expressed in <i>E. coli</i> and purified as membrane
fractions were immobilized in agarose matrixes on the oxygen sensing
layer. The activities of P450s were determined by evaluating the fluorescence
intensity enhancement of the oxygen sensor due to the oxygen consumption
by the metabolic reaction. By normalizing the responses with the amounts
of oxygen sensor and P450 enzymes in microwells, we could obtain fluorescence
enhancement patterns that were characteristic to the combination of
P450 isoforms and substrate material. The patterns obtained from two
psoralen derivatives resembled each other, whereas a structurally
different substrate (capsaicin) resulted in a distinct pattern. These
results suggest the potential of the microarray to analyze the activities
of diverse P450 isoforms in a high-throughput fashion. Furthermore,
mechanism-based inactivation (MBI) of P450 could be detected by successively
incubating a chip with different substrate solutions and measuring
the residual activities