CYP1B1 detection

Abstract

This unit describes procedures for measuring CYP1B1 gene expression by reverse transcription real-time PCR (qRT-PCR), CYP1B1 protein levels by western blotting, and CYP1B1 enzyme activity through conversion of 7-ethoxyresorufin substrate. To achieve specific measurement of CYP1B1 activity in the presence of CYP1A1 and CYP1A2, CYP1B1 inhibition and a subtractive approach have been adopted. 2,4,3',5'-Tetramethoxystilbene (TMS) is a potent and selective competitive inhibitor of CYP1B1 with an IC₅₀ of 3 nM for EROD and ~90 nM for E2 4-hydroxylation. Binding studies with purified CYP1B1 suggests that TMS interferes in the proximity of the heme region of CYP1B1 with high affinity. Compared to other potent inhibitors such as α-naphthoflavone, which is a known CYP1 family inhibitor with no selectivity between CYP1B1 and CYP1A2, TMS is ~50- and 520-fold selective for inhibition of CYP1B1 when compared to CYP1A1 and CYP1A2, respectively. Thus, TMS can serve as a helpful chemical scalpel for dissecting CYP1B1 activity from the overall activity of CYP1 family members against ethoxyresorufin.