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Research ArticleArticle

Measurement of Membrane-Bound Human Heme Oxygenase-1 Activity Using a Chemically Defined Assay System

Warren J. Huber III, Christopher C. Marohnic, Michelle Peters, Jawed Alam, James R. Reed, Bettie Sue Siler Masters and Wayne L. Backes
Drug Metabolism and Disposition April 2009, 37 (4) 857-864; DOI: https://doi.org/10.1124/dmd.108.025023
Warren J. Huber III
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Christopher C. Marohnic
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Michelle Peters
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Jawed Alam
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James R. Reed
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Bettie Sue Siler Masters
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Wayne L. Backes
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Abstract

Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH-cytochrome P450 reductase (CPR). Although most studies with HO used a soluble 30-kDa form, lacking the C-terminal membrane-binding region, recent reports show that the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity was elevated 5-fold, and the Km for CPR decreased approximately 50-fold. The goal of these studies was to accurately measure HO activity using a coupled assay containing purified biliverdin reductase (BVR). This allows measurement of bilirubin formation after incorporation of full-length CPR and heme oxygenase-1 (HO-1) into a membrane environment. When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2 to 3 min; however, the reaction was only linear for 10 to 30 s when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO-1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ml) and hBVR (0.025–0.05 μM), but neither supplement increased the time that the reaction remained linear. Various concentrations of superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4 to 5 min, even at high CPR levels. These results not only show that HO-1-generated hydrogen peroxide leads to a decrease in HO-1 activity but also provide for a chemically defined system to be used to examine the function of full-length HO-1 in a membrane environment.

Footnotes

  • This work was supported in part by the National Institutes of Health National Institute of Environmental Health Sciences [Grant ES004344]; the National Institutes of Health National Institute of General Medical Sciences [GM081568]; and the Robert A. Welch Foundation [AQ-0012] (to B.S.S.M.).

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

  • doi:10.1124/dmd.108.025023.

  • ABBREVIATIONS: HO-1, heme oxygenase-1, full-length; CO, carbon monoxide; Fe2+, ferrous iron; BVR, biliverdin reductase; P450, cytochrome P450; CPR, NADPH-cytochrome P450 reductase; ER, endoplasmic reticulum; hBVR, human biliverdin reductase; DLPC, dilauroylphosphatidylcholine; SOD, superoxide dismutase; H2O2, hydrogen peroxide; BSA, bovine serum albumin; RCS, reconstituted system; sHO-1, 30-kDa soluble human heme oxygenase-1.

    • Received October 7, 2008.
    • Accepted January 7, 2009.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 37 (4)
Drug Metabolism and Disposition
Vol. 37, Issue 4
1 Apr 2009
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Research ArticleArticle

Measurement of Membrane-Bound Human Heme Oxygenase-1 Activity Using a Chemically Defined Assay System

Warren J. Huber, Christopher C. Marohnic, Michelle Peters, Jawed Alam, James R. Reed, Bettie Sue Siler Masters and Wayne L. Backes
Drug Metabolism and Disposition April 1, 2009, 37 (4) 857-864; DOI: https://doi.org/10.1124/dmd.108.025023

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Research ArticleArticle

Measurement of Membrane-Bound Human Heme Oxygenase-1 Activity Using a Chemically Defined Assay System

Warren J. Huber, Christopher C. Marohnic, Michelle Peters, Jawed Alam, James R. Reed, Bettie Sue Siler Masters and Wayne L. Backes
Drug Metabolism and Disposition April 1, 2009, 37 (4) 857-864; DOI: https://doi.org/10.1124/dmd.108.025023
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