Assay of Heme Oxygenase‐1 through the Direct Measurement of Biliverdin Formation or Iron Release

Abstract

Heme oxygenase 1 (HO‐1) converts heme to CO, ferrous iron, and biliverdin (BV), receiving electrons used for catalysis from NADPH cytochrome P450 reductase (CPR). Enzymatic studies with the purified, 30 kDa form of HO‐1 routinely use a coupled assay containing biliverdin reductase (BVR) which converts BV to bilirubin (BR). BVR is believed to be required for optimal HO‐1 activity. The goal of this study was to determine if HO‐1 activity could be monitored directly by following BV generation or iron release (using the ferrous iron chelator, ferrozine) in the absence of BVR. Using assays for each of the three end‐products, we found that in the presence of catalase, HO‐1 activity was stimulated and comparable rates were measured in the assays. Absorbance scans revealed characteristic spectra for BR, BV and/or the ferrozene‐iron complex. The optimal conditions were slightly different for the direct and coupled assays. BSA activated the coupled but inhibited the direct assays, and the assays had different pH optima. By measuring the activity of BVR directly using BV as a substrate, these differences were attributed to different enzymatic properties of BVR and HO‐1. Thus, BVR is not needed to measure the activity of HO‐1 when catalase is present. In fact, the factors affecting catalysis by the HO‐1 are better understood using the direct assays because the coupled assay can be influenced by properties of BVR. (Supported by ES004344)