EXCHANGE OF SUBUNIT INTERFACES BETWEEN RECOMBINANT ADULT AND FETAL HEMOGLOBINS - EVIDENCE FOR A FUNCTIONAL INTERRELATIONSHIP AMONG REGIONS OF THE TETRAMER

The inter-relationship between the interior subunit interfaces and the exterior diphosphoglycerate (DPG) binding region of the hemoglobin te tramer and the effects of a specific N-terminal acetylation on tetrame r assembly have been evaluated, Tetrameric fetal hemoglobin F in the l iganded state was found to dissociate to dimers much less than previou sly appreciated, i.e, about 70 times less than adult hemoglobin A (K-d = 0.01 mu M and 0.68 mu 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 t he gamma-chains are acetylated, dissociates like HbA, To determine whe ther this feature of HbF could be transferred to hemoglobin A, the sin gle amino acid difference in their alpha(1) beta(2)/alpha(1) gamma(2) interfaces and the 4 amino acid differences in their alpha(1) beta(1)/ alpha(1) gamma(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 i soelectric focusing, the calculated amino acid composition, and normal circular dichroism properties, oxygen binding, and cooperativity, Alt hough 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 mu M) was between th at of HbA and HbF despite the fact that it was composed entirely of Hb F subunit interfaces, The results indicate that regions of the tetrame r 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 inte r-relationships in hemoglobin.