Human heme oxygenase-1 efficiently catabolizes heme in the absence of biliverdin reductase.
Heme oxygenase 1 (HO-1) uses molecular oxygen and electrons from NADPH cytochrome P450 reductase to convert heme to CO, ferrous iron, and biliverdin (BV). 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 whether 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 HO-1 activity was stimulated in the presence of catalase and comparable rates were measured with each assay. Absorbance scans revealed characteristic spectra for BR, BV, and/or the ferrozine-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 HO-1 are better understood using the direct assays because the coupled assay can be influenced by properties of BVR.
Reed
James R
JR
Department of Pharmacology and the Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA. rreed@lsuhsc.edu
Huber
Warren J
WJ
Backes
Wayne L
WL
D000818:Animals; D001663:Bilirubin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D001664:Biliverdine / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002374:Catalase / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D005297:Ferrozine / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D051547:Heme Oxygenase-1 / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits
D005297:Ferrozine; D006418:Heme; D002374:Catalase; D051547:Heme Oxygenase-1; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase; D009251:NADPH-Ferrihemoprotein Reductase; D001664:Biliverdine; D001663:Bilirubin