Sickle hemoglobin polymer stability probed by triple and quadruple mutant hybrids

As part of an effort to understand the interactions in HbS polymerization, we have produced and studied a recombinant triple mutant, D6A(alpha)/D75Y(alpha)/E121R(beta), and a quadruple mutant comprising the preceding mutation plus the natural genetic mutation of sickle hemoglobin, E6V(beta). These recombinant hemoglobins expressed in yeast were extensively characterized, and their structure and oxygen binding cooperativity were found to be normal. Their tetramer-dimer dissociation constants were within a factor of 2 of HbA and HbS. Polymerization of these mutants mixed with HbS was investigated by a micromethod based on volume exclusion by dextran. The elevated solubility of mixtures of HbS with HbA and HbF in dextran could be accurately predicted without any variable parameters. Relative to HbS, the copolymerization probability of the quadruple mutant/HbS hybrid was found to be 6.2, and the copolymerization probability for the triple mutant/HbS hybrid was 0.52. The pure quadruple mutant had a solubility slightly above that of its hybrid with HbS. One way to explain these results is to require significant cis-trans differences in the polymer and that HbA assemble above 42.5 g/dl. A second way to explain these data is by the modification of motional freedom, thereby changing vibrational entropy in the polymer.