SYSTEMATIC ENHANCEMENT OF POLYMERIZATION OF RECOMBINANT SICKLE HEMOGLOBIN

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In order to provide quantitative information on the sties that promote polymerization of sickle hemoglobin after formation of the initial hydrophobic bond involving Val-6(?) (E6V(?)) and also to provide hemoglobins with an enhanced polymerization that could be used in a mouse model for sickle cell amenia, we have expressed recombinant double, triple and quadrupole HbS mutants with substitutions on both the ?- and ?- chains, E6V(?)/E121R(?), D75Y(?)/E6V(?)/E121R(?) and D6A(?)D75Y(?)E6V(?)/ E121R(?). These recombinant hemoglobins were extensively characterized by HPLC analysis, SDS-PAGE, isoelectric focusing, amino acid analysis and mass spectroscopy. They retained the functional properties of the Hb cont. 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 (?D6A, ?D75Y and ?E121R) on the ?- and ??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 demonstates the feabilibility 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 published X. Liu, U.A. Mirza, B.T. Chait, J. M. Manning, Blood 90 (1997) 4620-4627.