LINKAGE BETWEEN OXYGENATION AND SUBUNIT ASSOCIATION IN HUMAN HEMOGLOBIN KANSAS - CONCENTRATION-DEPENDENCE OF THE OXYGEN BINDING EQUILIBRIA

The O2 binding equilibria of human Hb Kansas (a variant with a substitution at the .alpha.1.beta.2 interface) was measured as a function of protein concentration with an automated Imai apparatus and a Gill cell. Measurements were made at 20.degree. C in 0.05 M Tris/HCl buffer, pH 7.5, containing 0.1 M NaCl, 1 mM EDTA. The overall O2 affinity of the Hb decreased greatly when the concentration was increased from 0.36 .mu.M to 6.2 mM heme; the pressure of half-saturation increases from 4.7 to 32 mm Hg. The Hill coefficient at half-saturation was near 1.5 at all concentrations. These data were combined with an earlier determination of the dimer-tetramer association constant and analyzed with the linkage relations to yield changes in the free energies of the formation of the intersubunit contacts and of ligand binding for the dimer and tetramer at successive oxygenation steps. Only a small degree of cooperativity was manifested in the differences between the successive steps of O2 binding by Hb Kansas. The .beta. subunit, known to have an intrinsically low affinity for O2, appeared to contribute pseudo-negative cooperativity to the O2 binding by the dimers and the tetramers. A small positive contribution to the cooperativity occurred in the association equilibrium between the high affinity dimeric species and the low affinity tetrameric species.