Measurement of membrane-bound human heme oxygenase-1 activity using a chemically defined assay system

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 microM), 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.

Fig. 1.

Heme degradation pathway. After an obligatory interaction with CPR, HO-1 initiates the oxidative degradation of heme to biliverdin, CO, and Fe²⁺. Biliverdin is further metabolized to the lipophilic product, bilirubin, by BVR. Although HO-1 and CPR exist bound to the endoplasmic reticulum membrane, BVR is present in the cytosol.

Fig. 2.

Bilirubin formation catalyzed by BVR from two different sources. Recombinant hBVR was purified to apparent homogeneity, and rat liver cytosol served as a partially purified source of BVR. Real-time curves from 0 to 300 s of bilirubin formation catalyzed by partially pure BVR (solid line) and pure BVR (dashed line).

Fig. 3.

BSA is required for optimal bilirubin production at subsaturating and saturating CPR levels. A, BSA titration of a bilirubin assay at 1:5 CPR/HO-1 (□, dashed line) and 2:1 CPR/HO-1 (▴, solid line). B, real-time kinetic traces of bilirubin production with 0 mg/ml BSA, 1:5 CPR/HO-1 (——); 0 mg/ml BSA, 2:1 CPR/HO-1 (— ·· —); 0.25 mg/ml BSA, 1:5 CPR/HO-1 (— · —); and 0.25 mg/ml BSA, 2:1 CPR/HO-1 (- - -). HO-1 concentration was 0.05 μM for all the assays. Values in A represent the mean ± S.E. of n = 3.

Fig. 4.

BVR titration and real-time curves of bilirubin production. A, BVRs ranging from 0 to 0.1 μM were analyzed to determine the optimal concentration for bilirubin production at subsaturating and saturating CPR concentrations: 1:5 CPR/HO-1 (□) and 2:1 CPR/HO-1 (▴). B, real-time bilirubin assay curves of the four highest BVR concentrations (0.01–0.1 μM). HO-1 concentration was 0.05 μM for all the assays. Values in A represent the mean ± S.E. of n = 3.

Fig. 5.

Effect of SOD and catalase on the linearity of the bilirubin formation real-time activity curves. A and B, increasing amounts of superoxide dismutase (0, 0.00025, 0.00125, 0.00625, 0.025, 0.25, 0.625, and 1.25 U/μl) were added to the reaction mixture containing 1:5 (A) and 2:1 (B) CPR/HO-1. C and D, catalase (0, 0.00025, 0.00625, 0.025, 0.25, 0.625, 1.25, and 2.5 U/μl) was titrated into the 1:5 (C) and 2:1 (D) CPR/HO-1 reactions. HO-1 concentration was 0.05 μM for all the reactions.

Fig. 6.

Effect of H₂O₂ on HO-1 activity. H₂O₂ was added to a series of HO-1 reactions containing 2:1 CPR/HO-1 in the absence of catalase. H₂O₂ concentrations included 0 μM (▵), 5 μM (♦), 15 μM (○), 30 μM (▪), and 60 μM (⋄). Bilirubin formation in the presence of catalase and no exogenous H₂O₂ was included for comparison (dashed line). Each point represents the average of three determinations.

Fig. 7.

Generation of H₂O₂ by full-length and soluble HO-1. H₂O₂ produced by HO-1 and sHO-1 was measured at various CPR/HO-1 ratios. The HO-1/sHO-1 concentration was 0.5 μM. Low, mid, and high CPR corresponds to a DLPC/CPR reconstituted system (devoid of HO-1) using the same CPR concentrations as those used in the 1:5, 2:1, and 15:1 conditions. N.D. indicates that H₂O₂ was not detected. Each bar represents the mean ± S.E.M. for three determinations.

Fig. 8.

DLPC titration of HO-1 in RCS. DLPC levels were varied from 0 to 1200:1 DLPC/HO-1. After the 2-h incubation period at room temperature, the RCSs were assayed by monitoring bilirubin formation at 464 to 530 nm. Shown here are 1:5 CPR/HO-1 (▪) and 2:1 CPR/HO-1 (▵). The HO-1 concentration was 0.05 μM for all the assays. Values represent the mean ± S.E. of n = 3.

Fig. 9.

Effect of CPR concentration on HO-1 activity. A, time course for bilirubin formation. CPR concentrations included 0 μM (•), 0.0015 μM (○), 0.003 μM (▪), 0.00625 μM (□), 0.0125 μM (▴), 0.025 μM μM (▵), 0.05 μM (♦), and 0.1 μM (⋄). B, the rate of bilirubin formation as a function of CPR concentration. All the assays contained 0.05 μM HO-1. Values in B represent the mean ± S.E. of n = 3.