Functional studies and polymerization of recombinant hemoglobin Glu-alpha(2)beta(2)6(A3)->Val/Glu-7(A4)->Ala

In hemoglobin (Hb) S the hydrophobic mutated residue Val-beta 6(A3) (donor site) closely interacts with the hydrophobic side groups of Phe-beta 85(F1) and Leu-beta 88(F4) (EF pocket, acceptor site) of a neighboring tetramer, resulting in decreased solubility and polymerization of the deoxy-Hb. The beta 6(A3) residue is followed by two charged residues Glu-beta 7(A4) and Lys-beta 8(A5). This cluster has no attraction for the hydrophobic EF pocket. We have modified the beta 7(A4) residue next to the donor site Val-beta 6(A3), replacing the charged Glu by a hydrophobic Ala-(rHb beta E6V/E7A). The single mutant Glu-beta 7 --> Ala-(rHb beta E7A) was also engineered. Both rHbs exhibit a heat instability and an increased oxygen affinity compared to Hb A and Hb S. There was a concentration dependence of the ligand binding properties (1-300 mu M in heme) indicating an increased amount of dimers relative to Hb A. The deoxy form of rHb beta EGV/E7A polymerizes in vitro, with a decreased rate of polymer formation relative to Hb S, while the single mutant beta E7A does not polymerize in the same experimental conditions, The Glu-beta 7(A4) --> Ala substitution does not increase the hydrophobic interaction between donor and acceptor site. We speculate that the loss of the normal saline bridge between Glu-beta 7(A4) and Lys-beta 132(H10) leads to an increased flexibility of the A helix and may account for the difference of the polymerization for this Hb S mutant.