Abstract
The N-terminal 18-amino acid sequence of the beta-chain of hemoglobin, as far as the end of the A helix, has been replaced by the corresponding sequence of the gamma-chain of fetal hemoglobin with the remaining sequence of the beta-chain retained (helices B through H). The gamma-beta-chain had the correct mass, and its entire sequence was established by mass spectrometric analysis of its tryptic peptides; the alpha-chain also had the correct mass. This recombinant hemoglobin (named Hb Felix) retains cooperativity and has an oxygen affinity like that of HbA both in the presence and absence of the allosteric regulators, 2,3-diphosphoglycerate or chloride but differs from HbF in its 2,3-diphosphoglycerate response. However, Hb Felix has some features that resemble fetal hemoglobin, i.e. its significantly decreased tetramer-dimer dissociation and its circular dichroism spectrum, which measure the strength of the tetramer-dimer interface in the oxy conformation and its rearrangement to the deoxy conformation, respectively. Even though Hb Felix contains the HbA amino acids at its tetramer-dimer interface, which is located at a distance from the substitution sites, its interface properties resemble those of HbF. Therefore, the N-terminal sequence and not just those amino acids directly involved at the subunit interface contacts with alpha-chains must have a strong influence on this region of the molecule. The results reinforce the concept of fluid long range relationships among various parts of the hemoglobin tetramer (Dumoulin, A., Manning, L. R., Jenkins, W. T., Winslow, R. M., and Manning, J. M. (1997) J. Biol. Chem. 272, 31326-31332) and demonstrate the importance of the N-terminal sequence, especially in some mutant hemoglobins, in influencing its overall structure by affecting the relationship between helices.
Highlights
The N-terminal 18-amino acid sequence of the -chain of hemoglobin, as far as the end of the A helix, has been replaced by the corresponding sequence of the ␥-chain of fetal hemoglobin with the remaining sequence of the -chain retained
Hb Felix has some features that resemble fetal hemoglobin, i.e. its significantly decreased tetramerdimer dissociation and its circular dichroism spectrum, which measure the strength of the tetramer-dimer interface in the oxy conformation and its rearrangement to the deoxy conformation, respectively
The molecular mass of the ␥- subunit of Hb Felix was determined by ESI/MS to be 15,921.3 Ϯ 0.3, which is in excellent agreement with the calculated value of 15,921.3 (Fig. 2B)
Summary
The N-terminal 18-amino acid sequence of the -chain of hemoglobin, as far as the end of the A helix, has been replaced by the corresponding sequence of the ␥-chain of fetal hemoglobin with the remaining sequence of the -chain retained (helices B through H). Adult and fetal hemoglobins have several significant differences in their functional behavior at the quaternary level These differences include their responses to DPG1 binding, which affect their ability to carry O2 (4), the strengths of their tetramer-dimer interactions as recently reported (5), and the circular dichroism and absorption spectra of the aromatic amino acids in their polypeptide chains (6). These well defined and accurately measured properties in adult and fetal hemoglobins are due to unique subunit interactions in the ␣22 and the ␣2␥2 tetramer, respectively, that originate from the 39 amino acid substitutions between the - and ␥-chains. We have expressed a recombinant Hb containing the eight amino acid
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