UNIT 4.38 CYP1B1 Detection
Published Online: 1 FEB 2012
Copyright © 2011 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Toxicology
How to Cite
Divi, R. L., Luch, A., Verma, M. and Mahadevan, B. 2012. CYP1B1 Detection. Current Protocols in Toxicology. 51:4.38:4.31.1–4.31.26.
- Published Online: 1 FEB 2012
- Published Print: FEB 2012
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. Curr. Protoc. Toxicol. 51:4.38.1-4.38.26. © 2012 by John Wiley & Sons, Inc.
- cytochrome P4501B1;
- quantitative gene expression;
- CYP1B1 enzyme activity;
- western blotting;
- CYP1B1 inhibition