Dimer-monomer dissociation of human hemoglobin A.

Isolated 3H-labeled human hemoglobin alpha chains were incubated with unlabeled carbonmonoxyhemoglobin A for 72 h in 0.01 M potassium phosphate, pH 7.0, at 25 degrees C. Following separation of the free alpha chain monomers and Hb A (alpha 2 beta 2) tetramers by electrophoresis on cellulose acetate strips or by chromatography on DEAE-cellulose columns, analysis of radioactivity showed a slow transfer of 3H-labeled alpha chains into Hb A. Separation of the globin chains of the isolated [3H] Hb A on a CM-cellulose column in 8 M urea showed that the radioactivity was in structurally intact alpha chains. Gel filtration of a reaction mixture on Sephadex G-75 in the presence of 0.15 M NaCl showed that the 3H-labeled alpha-chains in Hb A were an integral part of, and not nonspecifically adsorbed to, the tetramer. These results indicate that the free 3H-labeled alpha chain monomers were incorporated into Hb A by exchange with pre-existing unlabeled alpha chain subunits according to the scheme, alpha 2 beta 2 in equilibrium 2 alpha beta in equilibrium 2 alpha + 2 beta. The rate constant for the dissociation of alpha beta dimers to alpha and beta monomers, the rate-limiting step in this exchange reaction, was 4.0 (+/- 0.7) X 10(-3) h-1.