The Role of Heme in the Synthesis and Assembly of Hemoglobin

Abstract

Abstract The incorporation of labeled amino acids into the α and β chains of hemoglobin has been studied in rabbit reticulocytes. Relative rates of synthesis were assessed by measurement of the specific activities of globin and of constituent α and β chains prepared from unpurified, ribosomefree hemolysates. The α:β specific activity ratio of approximately 1.0 indicated that labeled α and β chains were produced in nearly equal numbers. However, in hemoglobin which was purified from these hemolysates two features were noted that indirectly suggested the presence of a pool of α chains and a smaller pool of β chains: (a) the α:β specific activity ratio was less than 1.0, and (b) a loss of α chain radioactivity and a smaller loss of β chain radioactivity were observed following purification of the hemoglobin. More direct evidence for these previously formed pools has been found in (a) the radioactivity elution pattern observed on gel filtration of unpurified hemolysate, which revealed a radioactive protein of high specific activity, and (b) further analysis of this minor peak by ion exchange chromatography, peptide mapping, and spectrophotometry. Incubation with added hemin (1 x 10-4 m) was associated with (a) an increase in the specific activities of globin and of α and β chains, (b) the disappearance of the pool of β chains, which probably existed as αβ dimers, (c) persistence of a pool of α chains during the periods of incubation used, and (d) an increase in the α:β specific activity ratio in purified hemoglobin. A model of hemoglobin biosynthesis is suggested which could account for the observed effects of added hemin in terms of (a) stimulation of the synthesis of α and β chains, (b) combination of hemin with αβ dimers (globin) to form hemoglobin, and (c) promotion of the assembly of newly synthesized α and β chains, thus largely bypassing the pool of α chains. In this manner, heme may be said to coordinate as well as stimulate the synthesis of hemoglobin.