Three novel recombinant mutants of sickle hemoglobin (Hb S, β6Glu → Val) have been constructed to assess the role of proline at α114 and threonine at β87 in the polymerization of deoxygenated Hb S. Using the hemoglobin expression system (pHE2) designed in our laboratory, four plasmids were expressed separately in Escherichia coli to produce the four recombinant hemoglobins: r Hb S (β6Glu → Val); r Hb S-Chiapas (β6Glu → Val, α114Pro → Arg); r Hb S-D-Ibadan → (β6Glu → Val, β87Thr → Lys); and r Hb S-Chiapas-D-Ibadan (β6Glu → Val, α114Pro → Arg, β87Thr → Lys). The structural features of these four recombinant hemoglobins were analyzed by proton nuclear magnetic resonance spectroscopy, and were found to be similar to those of human normal adult hemoglobin (Hb A) under identical conditions. The recombinant hemoglobins were further investigated by measuring the oxygen-binding properties, which were found to be comparable to those of Hb A. Delay-time gelation studies of the three mutants of r Hb S were carried out in 1.8 M potassium phosphate (pH 7.34) by a temperature jump from 4°C to 30°C and an increase in delay time over that of r Hb S was observed, as well as an overall decrease in the polymerization of these three mutants of Hb S. A more detailed and quantitative investigation has also been carried out to determine the equilibrium solubility (C(sat)) in 0.1 M potassium phosphate (pH 7.35) at 25°C of the three Hb S mutants as well as of mixtures of these mutants with Hb S versus mixtures of fetal hemoglobin (Hb F) and Hb A with Hb S. The inhibition of polymerization demonstrated in these experiments suggests that the interactions involving the two amino acid residues α114Pro and β87Thr are very important to the formation of Hb S polymer, and modification of these amino acids results in an anti-sickling potential. Of particular interest is the inhibitory effect of α114Pro → Arg, which offers a novel opportunity to use an α-chain construct, in addition to a β-chain construct in the same vector, in gene therapy for sickle cell anemia, with the objective of modifying a larger number of hemoglobin tetramers at a given level of expression.
- Gene therapy
- Inhibition of Hb S polymerization
- Intermolecular contact sites in Hb S polymer
- Recombinant mutants of Hb S
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology