Purification and substrate specificity of bovine liver-ferrochelatase.
Bovine ferrochelatase from liver mitochondria was purified 1434-fold with a 31% yield to apparent homogeneity by a procedure involving solubilization, ammonium sulfate fractionation and blue Sepharose CL-6B chromatography. The molecular weight of the homogeneous protein was 42 500 when measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A molecular weight of approximately 200 000 was obtained by Sepharose 6B gel filtration. The specific activity for mesoheme synthesis was 413 nmol x mg protein-1 x min-1 at 37 degrees C and for protoheme synthesis 88 nmol x mg-1 x min-1. The optimum pH was 8.0 and Km values for the substrates were: protoporphyrin IX, 54 microM; mesoporphyrin IX, 46 microM; iron with protoporphyrin IX, 46 microM, iron with mesoporphyrin IX, 44 microM. The purified enzyme inserted iron into the following dicarboxylic porphyrins in descending order: meso-, deutero-, 2,4-diacetyldeutero-, hemato-, and protoporphyrin IX. This did not take place in the case of 2,4-diformyldeuteroporphyrin IX. Porphyrin c was converted to only a negligible amount of heme c, and coproporphyrin III did not act as a substrate at all. When metal specificity was examined, the highest value was obtained with zinc, decreasing in order with iron, cobalt and nickel. The enzyme failed to catalyze the insertion of copper or manganese into porphyrin. An antibody specific for the purified bovine ferrochelatase was prepared, and studies confirmed that the synthetic activities of iron-porphyrin, zinc-porphyrin and cobalt-porphyrin are ascribable to ferrochelatase.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D002417:Cattle; D005294:Ferrochelatase / Q000276:immunology / Q000302:isolation & purification*; D008190:Lyases / Q000302:isolation & purification*; D008670:Metals / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D011166:Porphyrins / Q000378:metabolism; D013379:Substrate Specificity
D000906:Antibodies; D008670:Metals; D011166:Porphyrins; D008190:Lyases; D005294:Ferrochelatase