%0 Journal Article %A Warren J. Huber III %A Christopher C. Marohnic %A Michelle Peters %A Jawed Alam %A James R. Reed %A Bettie Sue Siler Masters %A Wayne L. Backes %T Measurement of Membrane-Bound Human Heme Oxygenase-1 Activity Using a Chemically Defined Assay System %D 2009 %R 10.1124/dmd.108.025023 %J Drug Metabolism and Disposition %P 857-864 %V 37 %N 4 %X 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. The American Society for Pharmacology and Experimental Therapeutics %U https://dmd.aspetjournals.org/content/dmd/37/4/857.full.pdf