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Enzymatic Analysis of cDNA-Expressed Human CYP1A1, CYP1A2, and CYP1B1 With 7-Ethoxyresorufin as Substrate

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Cytochrome P450 Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 320))

Abstract

Cytochrome P450 (P450) enzymes belonging to the CYP1 family are highly inducible by polycyclic aromatic hydrocarbons and other environmental chemicals and play a major role in the metabolism of many foreign chemicals and endogenous substances. We describe a spectrofluorometric method for determining 7-ethoxyresorufin O-dealkylation catalyzed by CYP1A1, CYP1A2, and CYP1B1. The formation of the enzymatic product, resorufin, is monitored continuously by fluorescence using an excitation wavelength of 530 nm and an emission wavelength of 580 nm. This method can be applied to assay P450-catalyzed formation of resorufin from other alkoxyresorufins, such as 7-methoxyresorufin, 7-benzyloxyresorufin, and 7-pentoxyresorufin. It can also be used to assay 7-ethoxyresorufin O-dealkylation activity in isolated hepatocytes and cultured cells that express this P450 activity.

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Acknowledgments

This work was supported in part by the Canadian Institutes of Health Research (grant MOP-42385) and by the National Institutes of Health (grant 5 P42 ES07381).

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Authors and Affiliations

  1. Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada

    Thomas K. H. Chang

  2. Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, MA, USA

    David J. Waxman

Authors
  1. Thomas K. H. Chang
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  2. David J. Waxman
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Editor information

Editors and Affiliations

  1. School of Biological Sciences, Queen Mary University of London, London, UK

    Ian R. Phillips

  2. Department of Biochemistry and Molecular Biology, University College London, London, UK

    Elizabeth A. Shephard

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© 2006 Humana Press Inc.

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Chang, T.K.H., Waxman, D.J. (2006). Enzymatic Analysis of cDNA-Expressed Human CYP1A1, CYP1A2, and CYP1B1 With 7-Ethoxyresorufin as Substrate. In: Phillips, I.R., Shephard, E.A. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology, vol 320. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-998-2:85

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  • DOI: https://doi.org/10.1385/1-59259-998-2:85

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-58829-441-8

  • Online ISBN: 978-1-59259-998-1

  • eBook Packages: Springer Protocols

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