Autonomous synthesis of α and β hemoglobin chains in rabbit erythroid cells

Hemoglobin β-chain synthesis by rabbit erythroid cells was tested for dependence on availability of complementary α-chains. Reticulocytes and bone marrow cells were obtained from variant rabbits that have hemoglobin with isoleucine in α-chains but not in β-chains. This characteristic permits the use of L-o-methylthreonine, a specific isoleucine antagonist, to inhibit selectively the synthesis of hemoglobin α-chains without directly affecting that of β-chains. Study of hemoglobin synthesis by bone marrow cells presents two problems that require careful management: (A) the fragility of the globin-synthesizing apparatus and (B) the isolation of globin from the various proteins made by the mixture of nucleated cells. Disruption of synthetic activity was minimized by collecting the bone marrow in autologous plasma then removing fat and connective tissue while the cells were suspended in this medium. Purification involved gel filtration of hemoglobin and globin then CM-cellulose chromatography of globin chains. Absence of radioactive isoleucine in β-chains demonstrated the efficacy of this scheme in removing isoleucine-containing proteins that otherwise elute with β-chains on CM-cellulose columns. In reticulocytes, when synthesis of α-chains is inhibited by 30%-80%, that of β-chains is stimulated by 20%-60%, but in marrow cells, incorporation into β-chains stays at control level when α-incorporation is inhibited. The data indicate that β-chain synthesis is independent of the availability of complementary α-chains.