Reconstitution of the α and β subunits of human hemoglobin from separated α- and β-globin chains is described. The absorption and circular dichroism spectra, sedimentation coefficients, and sulfhydryl reactivities of the reconstituted subunits are compared with those of native subunits, which are regenerated from their p-mercuribenzoate chains by a novel procedure. The reconstituted subunits specifically recombine with the complementary native ones. Each property of the α subunit agrees, within experimental uncertainty, with the corresponding one of the subunit from which heme was never removed. Thus, markedly disordered and aggregated α-globin is restored to the highly ordered, monomeric α subunit upon recombination with 1 eq of hemin or hemin dicyanide. The reconstituted β subunit also compares well with the native subunit, except for a minor difference in a portion of the near-ultraviolet circular dichroism spectrum and a diminished sulfhydryl reactivity. Possible reasons for these differences are discussed. In the met form neither reconstituted subunit forms hemochromogen for several days, whereas the original met subunits become denatured within hours, probably owing to the necessity of exposing the latter, but not the former, to ferricyanide ion in producing the met forms.

It is shown that while α- and β-globins do not combine at 4° (Yip, Y. K., Waks, M., and Beychok, S. (1972) J. Biol. Chem. 247, 7237), recombination does occur if either chain contains heme. The recombination produces half-filled (HF) molecules which are of two kinds, depending on which chain contains heme. The moleclar weights of these molecules are different. HFα, the intermediate prepared by mixing α subunits with β-globin, is a dimer; HFβ, the intermediate containing heme on its β chains, is a tetramer. No interchange of heme among chains occurs in the cyanmet forms in either molecule. In each, moreover, the conformation of the heme-free chain differs from that of the same chain when unbound. Both HF molecules bind 1 eq of heme, producing tetrameric hemoglobin.

The most dramatic finding in this work is that the disordered α-globin can be refolded not only by binding heme but also by combining with a β subunit, although the refolding in the latter case is not as complete as in the former.

These results show that the primary sequences of the globin chains generate unstable conformations, which are stabilized by combination with heme. They demonstrate also that refolding of a polypeptide chain can be induced by interaction with a neighboring subunit in an assembly process.