Sickle Cell Hemoglobin with Mutation at αHis-50 Has Improved Solubility

Background: Deoxy sickle cell hemoglobin (Hb S) tetramers polymerize in solution via lateral and axial contacts among neighbors. Results: His-50 Gln mutation can, whereas His-20 Gln mutation cannot, improve the solubility of Hb S. Conclusion: (2)His-50 interacts with the (S)(1) CD corner, whereas (2)His-20 has minimal interaction with (S)(1)Glu-22. Significance: (2)His-50 contributes significantly to the polymerization of the Hb S tetramers.The unliganded tetrameric Hb S has axial and lateral contacts with neighbors and can polymerize in solution. Novel recombinants of Hb S with single amino acid substitutions at the putative axial (recombinant Hb (rHb) (E6V/H20R) and rHb (E6V/H20Q)) or lateral (rHb (E6V/H50Q)) or double amino acid substitutions at both the putative axial and lateral (rHb (E6V/H20R/H50Q) and rHb (E6V/H20Q/H50Q)) contact sites were expressed in Escherichia coli and purified for structural and functional studies. The H-1 NMR spectra of the CO and deoxy forms of these mutants indicate that substitutions at either His-20 or His-50 do not change the subunit interfaces or the heme pockets of the proteins. The double mutants show only slight structural alteration in the -heme pockets. All mutants have similar cooperativity (n(50)), alkaline Bohr effect, and autoxidation rate as Hb S. The oxygen binding affinity (P-50) of the single mutants is comparable with that of Hb S. The double mutants bind oxygen with slightly higher affinity than Hb S under the acidic conditions. In high salt, rHb (E6V/H20R) is the only mutant that has a shorter delay time of polymerization and forms polymers more readily than Hb S with a dextran-Csat value of 1.86 +/- 0.20 g/dl. Hb S, rHb (E6V/H20Q), rHb (E6V/H50Q), rHb (E6V/H20R/H50Q), and rHb (E6V/H20Q/H50Q) have dextran-Csat values of 2.95 +/- 0.10, 3.04 +/- 0.17, 11.78 +/- 0.59, 7.11 +/- 0.66, and 10.89 +/- 0.83 g/dl, respectively. rHb (E6V/H20Q/H50Q) is even more stable than Hb S under elevated temperature (60 degrees C).