Abstract
Microsomal heme oxygenase has been purified from bovine spleen to homogeneity using DEAE-cellulose chromatography, initial hydroxyapatite chromatography, gel filtration, and repeat hydroxyapatite chromatography. The purified enzyme showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of congruent to 31,000. It had a Km for heme of 0.93 microM, a specific activity of 6770 units/mg of protein, and a turnover number of 3.5 mol/mol of enzyme/min. Amino acid analysis of the purified enzyme revealed an abundance of glutamine and glutamic acid residues, a log tryptophan content (2 tryptophan residues/mol of enzyme), and four free sulfhydryl groups, only two of which were accessible to sulfhydryl (--SH) inactivating reagents without unfolding of the protein. Consistent with these findings, the purified enzyme had a relatively low extinction coefficient at 280 nm (E11%cm = 8.12) and was relatively resistant to inactivation by --SH inactivating reagents. NADPH-cytochrome c reductase from bovine liver was purified to homogeneity and biliverdin reductase from bovine spleen was partially purified. The heme oxygenase system was reconstituted from preparations of all three purified enzymes and, utilizing this reconstituted system, the specific sites of the inhibitory actions of --SH inactivating reagents, inorganic metals, and metalloporphyrins on the heme degrading sequence of reactions were examined. Sn-, Co-, Zn-, and Mn-protoporphyrin strongly inhibited heme degradation in a competitive manner. The Ki values for Sn-, Co-, and Zn-protoporphyrin were determined to be 0;033, 0.082, and 0.13 microM, respectively. Mg-, Ni-, and Cu-protoporphyrin had little effect on heme degradation by the reconstituted system. Metals such as Pt2+ and Hg2+ strongly inhibited the activity of the reconstituted heme oxygenase system, but the principal site of action of these metals was at the level of NADPH-cytochrome c reductase or biliverdin reductase. Similarly --SH inactivating reagents, such as p-chloromercuribenzoate, 5,5'-dithiobis-(2-nitrobenzoate), or N-ethylmaleimide inhibited the reaction catalyzed by the reconstituted heme oxygenase system principally by inhibiting the activity of NADPH-cytochrome c reductase.
Highlights
Microsomal heme oxygenase has been purified from phyrin IX, to yieldbiliverdin IX, in an oxidative reaction bovine spleen to homogeneity using DEAE-cellulose requiring NADPH and molecular oxygen [1,2,3,4,5,6]
The heme oxygen- purified to homogeneity for the first time from bovine liver ase system was reconstituted from preparations of all and bovine spleen biliverdin reductase was partially purified
Metals such as p t z ’ and H 6 + strongly inhibited the activity of the reconstituted heme oxygenase system, but the principal site of action of these metals was at the level of NADPH-cytochrome c reductase orbiliverdin reductase
Summary
Three purified enzymes and, utilizing this reconstituted system, the specific sites of the inhibitory actions of ”SH inactivating reagents, inorganic metals, and metalloporphyrins on the heme degrading sequence of reactions were examined. Mg-, Ni-, and Cu-protoporphyrin had little effect on heme degradation by the reconsti- Utilizing these purified enzymes in a reconstituted heme oxidation system, it was possibleto examine the specific sites of action of compounds such as sulfhydryl reagents, inorganic metals, and metalloporphyrins which are known to inhibit heme oxidation in microsomal preparations. The results of these experiments with the reconstituted heme oxygenase system indicate that thedegradation of heme to bile pigment is inhibited by such agents at different steps in the reaction sequence. Metals such as p t z ’ and H 6 + strongly inhibited the activity of the reconstituted heme oxygen-
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