A precise and rapid procedure employing gel filtration on Superose-12 to measure the tetramer-dimer dissociation constants of some natural and recombinant hemoglobins in the oxy conformation is described. Natural sickle hemoglobin was chosen to verify the validity of the results by comparing the values with those reported using an independent method not based on gel filtration. Recombinant sickle hemoglobin, as well as a sickle double mutant with a substitution at the Val-6(beta) receptor site, had approximately the same dissociation constant as natural sickle hemoglobin. Of the two recombinant hemoglobins with amino acid replacements in the alpha 1 beta 2 subunit interface, one was found to be extensively dissociated and the other completely dissociated. In addition, the absence of an effect of the allosteric regulators DPG and IHP on the dissociation constant was demonstrated. Thus, a tetramer dissociation constant can now be determined readily and used together with other criteria for characterization of hemoglobins and their interaction with small regulatory molecules.

The inter-relationship between the interior subunit interfaces and the exterior diphosphoglycerate (DPG) binding region of the hemoglobin tetramer and the effects of a specific N-terminal acetylation on tetramer assembly have been evaluated. Tetrameric fetal hemoglobin F in the liganded state was found to dissociate to dimers much less than previously appreciated, i.e. about 70 times less than adult hemoglobin A (K d ‫؍‬ 0.01 M and 0.68 M, for HbF and HbA, at pH 7.5, respectively) over the pH range 6.2-7.5, whereas HbF 1 , in which the N termini of the ␥-chains are acetylated, dissociates like HbA. To determine whether this feature of HbF could be transferred to hemoglobin A, the single amino acid difference in their ␣ 1 ␤ 2 /␣ 1 ␥ 2 interfaces and the 4 amino acid differences in their ␣ 1 ␤ 1 /␣ 1 ␥ 1 interfaces have been substituted in HbA to those in HbF. This pentasubstituted recombinant HbA/F had the correct molecular weight as determined by mass spectrometry, the expected mobility on isoelectric focusing, the calculated amino acid composition, and normal circular dichroism properties, oxygen binding, and cooperativity. Although HbA/F has the same amino acid side chains that bind DPG as HbA, its diminished response to 2,3-DPG resembled that of HbF. However, its tetramer-dimer dissociation constant (K d ‫؍‬ 0.14 M) was between that of HbA and HbF despite the fact that it was composed entirely of HbF subunit interfaces. The results indicate that regions of the tetramer distant from the tetramer-dimer interface influence its dissociation and, reciprocally, that the interfaces affect regions involved in the binding of allosteric regulators, suggesting flexible long range inter-relationships in hemoglobin.

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