The experiments described in this communication demonstrate that C14-tagged protoporphyrin 9 can be incorporated into the heme during the biosynthesis of hemoglobin.
1. In vitro observations:
(a) C14 protoporphyrin 9 was found to be incorporated into heme by hemolysates of chicken and human blood incubated at 37 C. The degree of incorporation by washed chicken erythrocytes was less, presumably because the protoporphyrin was not readily transferred across the cell membrane. Incorporation by hemolysates was inhibited completely at 1 x 10-2 M KCN at 4 C., markedly by 1 x 10-2 M KCN at 37 C. and partially by 1 x 10-3 M Pb at 37 C.
(b) The degree of incorporation was reduced by the addition of an equivalent quantity of delta-aminolevulinic acid. Furthermore, the incorporation of glycine-2-C14 into heme was reduced by the addition of an equivalent quantity of protoporphyrin 9.
2. In vivo observations:
Intravenously administered C14 protoporphyrin was incorporated into the circulating hemoglobin of two rabbits with a phenylhydrazine-induced hemolytic anemia.
These observations provide support for the view that protoporphyrin 9 itself is a true direct precursor of hemoglobin, in the biosynthetic pathway between porphobilinogen and heme. Comparative studies of rates of incorporation of C14 protoporphyrin 9 and its precursors into heme in vitro may provide a useful tool for the study of heme synthesis in normal and pathologic conditions. For instance, it was shown that hemolysates from the blood of patients with thalassemia major, with poor iron and glycine utilization, rapidly incorporated the tagged protoporphyrin into heme.