Assembly of human adult and sickle hemoglobins from their oxygenated subunits. Differential rates of beta chain tetramer dissociation.

The rate of assembly of oxyhemoglobins A and S at pH 7 and 20 degrees C has been followed spectrophotometrically at 582.5 nm, a maximum of the difference spectrum of isolated oxygenated subunits and intact oxyhemoglobin. In 0.1 M potassium phosphate buffer, the resultant time courses following mixing of equivalent concentrations of alpha and beta chains were homogeneous and followed first order kinetics. These time courses were protein concentration independent over a range of 8.0 to 60 X 10(-6) M in heme after mixing and presumably reflect the rate of dissociation of the beta chain tetramer. This rate-limiting dissociation reaction was found to be nearly 2-fold slower for the beta S than the beta A chain tetramer exhibiting half-times of 27 and 15 min, respectively. The overall rate of formation of Hb S and Hb A appears to be significantly influenced by the rates of dissociation of their respective beta chains. These findings are in contrast to mixing experiments done in 0.01 M potassium phosphate buffer which revealed time courses which were heterogeneous and markedly dependent upon protein concentration. The stability of the oxygenated beta chain tetramer and, therefore, the overall kinetic profile of liganded hemoglobin reconstitution is acutely sensitive to buffer conditions.