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Volume 272, Number 50, Issue of December 12, 1997 pp. 31326-31332

Exchange of Subunit Interfaces between Recombinant Adult and Fetal Hemoglobins
EVIDENCE FOR A FUNCTIONAL INTER-RELATIONSHIP AMONG REGIONS OF THE TETRAMER

(Received for publication, June 26, 1997, and in revised form, October 1, 1997)

Antoine Dumoulin , Lois R. Manning , W. Terry Jenkins ^§ , Robert M. Winslow ^¶ and James M. Manning

From the  Department of Biology, Northeastern University, Boston, Massachusetts 02115, the ^§ Department of Chemistry, Indiana University, Bloomington, Indiana 47405, and the ^¶ Department of Medicine, Veterans Affairs Medical Center, University of California, San Diego, California 92161

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 µM and 0.68 µM, for HbF and HbA, at pH 7.5, respectively) over the pH range 6.2-7.5, whereas HbF₁, 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 ₁₂/₁₂ interfaces and the 4 amino acid differences in their ₁₁/₁₁ 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 µM) 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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