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|Title:||FUNCTIONAL SCREENING OF CYTOCHROME P450 ACTIVITY AND UNCOUPLING BY CAPILLARY ELECTROPHORESIS|
|Authors:||Harskamp, James G.|
|Advisor:||Wilson, Joanna Y|
|Keywords:||cytochrome p450;Uncoupling;capillary electrophoresis;reactive oxygen species;enzyme kinetics;Biology;Biology|
|Abstract:||<p>Cytochrome P450s are a super-family of heme containing proteins that are found in all domains of life and are involved in the synthesis and breakdown of steroids, xenobiotics, and pharmaceuticals. Using five heterologously expressed zebrafish (Danio rerio) CYP1s, an assay was developed for CYP activity in order to monitor the consumption of the cofactor NADPH, providing a label-free screening tool to determine function of novel CYP genes. Using well-established fluorogenic substrates, NADPH and NADP+ were separated by capillary electrophoresis (CE) from stopped CYP1 reactions and measured with UV absorbance detection as a surrogate to assess the rate of substrate metabolism. Product formation was confirmed by fluorometric detection of metabolites, giving rates of enzyme activity which could be compared to the rates of cofactor turn-over measured by CE. 17β-estradiol, four alkoxyresorufin and two coumarin based synthetic fluorogenic CYP substrates were screened for activity with recombinant zebrafish CYP1A, 1B1, 1C2, 1C2 and 1D1. Cofactor consumption was generally much larger than product formation for the majority of substrates and CYP1 isoforms, suggesting that the majority of metabolic events were uncoupled. Large uncoupling was seen in CYP1 when metabolizing estradiol, showing that endogenous compounds can also show severe uncoupling. Reactive oxygen species (ROS), a product of uncoupled events, were detected with 2,7- dichorofluorescein. Attempts for concomitant detection of ROS production and cofactor consumption with CE-UV detection were investigated, however, detection limits for 2,7-dichlorofluorescein were not adequate for detection of hydrogen peroxide production from CYP1 mediated reactions. Future work will be required to develop a single assay to quantitatively measure CYP activity by CE for functional determination of CYPs with unknown function.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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