Unequal Synthesis of Complementary Globin Chains of Human Fetal Hemoglobin by the Effect of L-O-Methylthreonine

Incorporation of radioactive amino acids into the α, β, and γ chains of human hemoglobin was studied with erythroid cells from newborn infants. When the cells were incubated in isoleucine-free medium together with l-O-methylthreonine, which is an antagonist of l-isoleucine, inhibition of γ chain synthesis occurred. The concomitant synthesis of α and β chains, which do not contain isoleucine, was not significantly affected. Inhibition of γ chain synthesis by as much as 90% had virtually no effect on the synthesis of complementary α chains, suggesting that the synthesis or release of α chains does not require prior synthesis of these non-α chains. As a result of the selective inhibition of synthesis of γ chains, cells incubated with the isoleucine antagonist synthesized a 2- to 3-fold excess of α chains over the total non-α chains. A major portion of the excess α chains was present in the cell lysates uncombined with hemoglobin, and had Sephadex gel filtration properties consistent with their being present as α dimers. When βa subunits were added to the lysates prior to Sephadex gel filtration, the excess α chain radioactivity appeared in the hemoglobin fraction, suggesting that the free α chains existed as completed globin-heme subunits which retained the capacity to combine with complementary subunits to form hemoglobin. When cells were incubated with l-O-methylthreonine and a radioactive amino acid in label-chase experiments, followed by 24-hour incubations, the excess radioactive α chains remained in the soluble cell fraction. This was in contrast with reported studies with β-thalassemia cells in which α chain precipitation was demonstrated during cell incubations in vitro. This difference appeared to be the result of intracellular exchange of the radioactive α chains with α chains of preexisting hemoglobin in the cells incubated with l-O-methylthreonine, a reaction which may be undetectable in β-thalassemia cells because of the presence of a preexisting pool of nonradioactive α chains in these cells.