Evidence for the presence of free and protein-bound nonhemoglobin heme in rabbit reticulocytes

Abstract

In the hemolysate of reticulocytes incubated with 59Fe or [2– 14C]glycine a radioactivity is found also in nonhemoglobin heme. About 96% of the radioactivity of nonhemoglobin heme is associated with proteins and approx. 4% is in a free form. Labeled free heme is removed from the reticulocyte hemolysate on the column of Sephadex G-25. Retained heme is eluted from the column of Sephadex G-25 by means of the special eluting solution containing serum albumin. Protein-bound nonhemoglobin heme radioactivity is recovered in hermin isolated from nonhemoglobin proteins separated by means of the chromatography on CM-Sephadex. Isonicotinic acid hydrazide which specifically inhibits heme synthesis prevents the incorporation of 59Fe into hemoglobin heme. In the hemolysate of cycloheximide incubated reticulocytes the radioactivity in both free and protein-bound nonhemoglobin heme increases essentially. The accumulation of 59Fe-labeled heme in the nonhemoglobin proteins in reticulocytes incubated with cycloheximide provides evidence for an existence of an increased pool of heme which is not incorporated into hemoglobin or catalase. The finding of the existence of free heme in reticulocytes might contribute to recent theories of regulation of hemoglobin synthesis.