Exchange of subunit interfaces between recombinant adult and fetal hemoglobins. Evidence for a functional inter-relationship among regions of the tetramer.
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 (Kd = 0.01 microM and 0.68 microM, 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 gamma-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 alpha1beta2/alpha1gamma2 interfaces and the 4 amino acid differences in their alpha1beta1/alpha1gamma1 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 (Kd = 0.14 microM) 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.
Dumoulin
A
A
Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA.
Manning
L R
LR
Jenkins
W T
WT
Winslow
R M
RM
Manning
J M
JM
D019794:2,3-Diphosphoglycerate / Q000494:pharmacology; D000328:Adult; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019281:Dimerization; D005319:Fetal Hemoglobin / Q000737:chemistry*; D006441:Hemoglobin A / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
D000596:Amino Acids; D011994:Recombinant Proteins; D019794:2,3-Diphosphoglycerate; D006441:Hemoglobin A; D005319:Fetal Hemoglobin