Premium
Measurement of Membrane‐Bound Human Heme Oxygenase‐1 Activity Using a Chemically Defined Assay System
Author(s) -
Huber Warren,
Marohnic Christopher,
Peters Michelle,
Alam Jawed,
Masters Bettie Sue S,
Backes Wayne L
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.751.2
Subject(s) - heme oxygenase , chemistry , heme , oxygenase , biliverdin reductase , catalase , reductase , bilirubin , biochemistry , enzyme , biology , endocrinology
Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH‐cytochrome P450 reductase (CPR). Although most studies with HO used a 30‐kDa form, lacking the C‐terminal membrane binding region, the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity being elevated 5‐fold, and the K m for CPR decreased approximately 50‐fold. The goal of these studies was to accurately measure membrane‐bound heme oxygenase‐1 (HO‐1) activity using a coupled assay containing purified biliverdin reductase (BVR). When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2‐3 min; however, the reaction was only linear for 10‐30 sec 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. Superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4‐5 minutes, even at high CPR levels. These results not only demonstrate that hydrogen peroxide leads to a decrease in HO‐1 activity, but also provide a chemically defined system to further examine the function of full‐length HO‐1 in a membrane environment.