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, Wayne L. Backes

Research output: Contribution to journalArticle

19 Scopus citations

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 K m 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.

Original languageEnglish (US)
Pages (from-to)857-864
Number of pages8
JournalDrug Metabolism and Disposition
Volume37
Issue number4
DOIs
StatePublished - Apr 2009

ASJC Scopus subject areas

  • Pharmacology
  • Pharmaceutical Science

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    Huber, W. J., Marohnic, C. C., Peters, M., Alam, J., Reed, J. R., Masters, B. S. S., & Backes, W. L. (2009). Measurement of membrane-bound human heme oxygenase-1 activity using a chemically defined assay system. Drug Metabolism and Disposition, 37(4), 857-864. https://doi.org/10.1124/dmd.108.025023