Purification of bovine protoporphyrinogen oxidase: immunological cross-reactivity and structural relationship to ferrochelatase.
Protoporphyrinogen oxidase, the penultimate enzyme in the haem biosynthetic pathway has been purified to apparent homogeneity from bovine liver mitochondria, by a published method (Dailey, H.A. and Fleming, J.E., (1983)), with an additional ion-exchange chromatography step, using a Mono Q column on an FPLC-system. This gave a product with a 68% yield and 870-fold purification. Protoporphyrinogen oxidase (EC 1.3.3.4) has an apparent Mr of 57,000 and the Km for protoporphyrinogen IX was 16.6 microM. Activity of the isolated enzyme was increased by 66% in the presence of oleic acid, and evidence was obtained for a FAD prosthetic group. Ferrochelatase (EC 4.99.1.1) was purified and antibodies were raised in rabbits against ferrochelatase and protoporphyrinogen oxidase, respectively. Anti-protoporphyrinogen oxidase IgG showed marked cross-reactivity with ferrochelatase and anti-ferrochelatase IgG cross-reacted with protoporphyrinogen oxidase. In addition, radiolabelled peptides of both enzymes, generated by chymotrypsin, demonstrated common peptides when analysed by two-dimensional chromatography.
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin; D003429:Cross Reactions; D000939:Epitopes; D005294:Ferrochelatase / Q000032:analysis* / Q000276:immunology; D007700:Kinetics; D008190:Lyases / Q000032:analysis*; D008930:Mitochondria, Liver / Q000201:enzymology; D010088:Oxidoreductases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010449:Peptide Mapping; D050768:Protoporphyrinogen Oxidase; D013057:Spectrum Analysis
D000939:Epitopes; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; D002918:Chymotrypsin; D008190:Lyases; D005294:Ferrochelatase