In order to provide quantitative information on the sites that promote polymerization of sickle hemoglobin after formation of the initial hydrophobic bond involving Val-6(p) (E6V(p)) and also to provide hemoglobins with an enhanced polymerization that could be used in a mouse model for sickle cell anemia, we have expressed recombinant double, triple and quadrupole HbS mutants with substitutions on both the a- and 0- chains, E6V(P)/EI2IR(P), D75Y(a)/E6V(P)/EI21R(p) and D6A(a)/D75Y(a)/E6V(0)/EI2IR(P). These recombinant hemoglobins were extensively characterized by HPLCanalysis, SDS-PAGE, isoelectric focusing, amino acid analysis and mass spectroscopy. They retained the functional properties of the Hb tetramer and polymerize in a linear manner at progressively lower Hb concentration as a function of the degree of substitution, suggesting that these remote sites (aD6A, aD75Y and PE121R) on the a- and 0-chains exhibit additive, enhanced polymerization properties. The quadrupole mutant has a polymerization concentration close to that of the purified SAD hemoglobin from transgenic mouse red cells consisting of HbS , Hb A ntilles and Hb D -Punjab. Normal mouse Hb increases the polymerization concentration of each mutant. Thus, the general approach of using recombinant hemoglobins as described here should prove useful in elucidating the quantitative aspects of the mechanism of HbS polymerization and in identifying the contribution of individual sites to the overall process. The strategy described here demonstrates the feasibility of a systematic approach to achieve future recombinant HbS mutants which could provide a new generation of the transgenic mouse model for sickle cell anemia. A paper describing this work has been submitted.
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