PURIFICATION AND CHARACTERIZATION OF HEME OXYGENASE FROM CHICK LIVER - COMPARISON OF THE AVIAN AND MAMMALIAN ENZYMES

A major inducible form of heme oxygenase (EC 1.14.99.3) was purified from liver microsomes of chicks pretreated with cadmium chlride. The purification involved solubilization of microsomes with Emulgen 913 and sodium cholate, followed by DEAE‐Sephacel, carboxymethyl‐cellulose (CM‐52) and hydroxyapatite chromatography, and FPLC through Superose 6 and 12 columns operating in series. The final product gave a single band on silver‐stained SDS/polyacrylamide gels (Mr= 33000). Optimal conditions for measurement of activity of solubilized heme oxygenase were studied. In a reconstituted system containing purified heme oxygenase, NADPH‐cytochrome reductase, biliverdin reductase and NADPH, the Km for free heme was 3.8 ± 0.5 μM; for heme in the presence of bovine serum albumin (5 mol heme/3mol albumin) the Km the 5.0 ± 0.8 μM; and the kmfor NADPH was 6.1 ± 0.4 μM (all values mean ± SD,n= 3). Oxygen concentration as low as 15üM, with saturating concentrations of heme and NADPH, did not affect the reaction rate, indicating that the supply of oxygen is not involved in the physiological regulation of activity of the enzyme. The pH optimum of the reaction was 7.4; at 37°C, the apparent Vmax was 580 ± 44 nmol biliverdin. (mg protein)−1· min−1 and the molecular activity was 19.2 min−1. Biliverdin IXa was the sole biliverdin isomer formed. In the presence of purified biliverdin reductase, biliverdin was converted quantitatively to bilirubin. Addition of catalase to the reconstituted system decreased the breakdown of heme to non‐biliverdin products and led to nearly stoichiometric conversion of heme to biliverdin. Activity of the enzyme in the reconstituted system was inhibited by metalloporphyrins in the following order of decreasing potency: tin mesoporphyrin > tin protoporphyrin > zine protoporphyrin > manganese protoporphyrin > cobalt protoporhyrin. Protoporphyrin (3.3 or 6.6 μM) (and several other porphyrins) and metallic ions (100 μM) alone had little if any inhibitory effect, except for Hg2+ which inhibited by 67% at 10 μM and totally at 15 μM. Following partial cleavage, fragments of the purified enzyme were sequenced. Comparison of sequences to those derived from cDNA sequences for the major inducible rat and human heme oxygenase showed 69% and 76% similarities, respectively. The histidine residue at position 132 of rat heme oxygenase‐1 and the residues (Lys128‐Arg136) flanking His132 were conserved in all three enzymes, as well as in the corresponding portion of a fourth less highly similar rat enzyme, heme oxygenase‐2. It is suggested that this region plays a critical role in heme binding and in catalyzing the heme oxygenase reaction. Copyright © 1990, Wiley Blackwell. All rights reserved