Immunochemical studies of ferrochelatase protein: characterization of the normal and mutant protein in bovine and human protoporphyria. Protoporphyria is a hereditary disorder characterized by a marked decrease in the activity of ferrochelatase, the terminal enzyme in the heme biosynthetic pathway. We have prepared specific polyvalent antibodies against bovine ferrochelatase in rabbits. The specificity of the antibody preparation against ferrochelatase was demonstrated by western blot analysis and immunoprecipitation of ferrochelatase activity. The antibody also cross-reacted weakly with ferrochelatase from human mitochondria. To quantify immunoreactive ferrochelatase in tissue samples, a kinetic-based enzyme-linked immunosorbent assay (k-ELISA) was developed. Ferrochelatase activity and the level of immunoreactive protein were measured in hepatic mitochondria isolated from six normal and nine protoporphyric (homozygous) cattle. Ferrochelatase activity was less than 10% of normal in mitochondria from protoporphyric animals; the amount of immunoreactive material was equivalent to that from normal animals. Similar studies were performed with samples from three normal and two protoporphyric (heterozygous) humans. Ferrochelatase activity was decreased in protoporphyric samples (about 17% of normal, but there was no concomitant decrease in immunoreactive material. These data demonstrate that a normal amount of ferrochelatase protein is present and suggest that bovine and human protoporphyria result from point mutations in the gene encoding ferrochelatase. 