High-Throughput Functional Screening of Steroid Substrates with Wild-Type and Chimeric P450 Enzymes
Flowchart for HT activity analysis. Upper left panel, LC/MS screenshot of the overlay of six chromatograms observed for CYP1A1-catalyzed hydroxylation of a substrate of at different incubation times (hydroxylated substrate [M + H] + = 253). Upper right panel, area of the metabolite peak at min versus incubation time. The four lower panels present different types of observed kinetics and rejection cases. Left panel, the case is kept for further analysis (metabolite production is linear over time).