Reductive hydroxyethylation of the α-amino groups of amidated hemoglobin S

Val-6(β) of hemoglobin S forms the primary site of intertetrameric interaction in the polymerization of deoxy hemoglobin S. However, a number of other intermolecular interactions contribute significantly to the polymerization process as well as to the stability of the polymerized gel. The strong stabilizing influence of Val-6(β) in the polymerization process is reflected in the fact that although a number of mutations at any one of the intermolecular contact regions (or perturbation of these contact regions by chemical modification) result in some increase in the solubility of deoxy hemoglobin S, none of these mutations and/or chemical modifications completely neutralize the polymerizing influence of Val-6(β), i.e., restores the solubility to that of hemoglobin A. Additivity and/or synergy of the solubilizing influence of two or more chemical modification reactions each of which independently increases the solubility may be considered as a possible strategy to restore the solubility of deoxy hemoglobin S to that of hemoglobin A. In the present study, the cumulative solubilizing influence of amidation of Glu-43(β) and hydroxyethylation of α-amino groups of hemoglobin S has been investigated by preparing hemoglobin S with double modification. Modification of Glu-43(β) by amidation with glycine ethyl ester did not influence the reactivity of the α-amino groups of hemoglobin S toward reductive hydroxyethylation, thus permitting the preparation of hemoglobin S with the two modifications. The reductive hydroxyethylation increased the oxygen affinity of amidated hemoglobin S to nearly the same degree as it does on modification of unmodified hemoglobin. In addition, hemoglobin S with double modification has a Hill coefficient that is the same as that of unmodified hemoglobin S, suggesting that the overall quaternary interaction of hemoglobin S with a double modification is nearly the same as the unmodified protein. However, the reductive hydroxyethylation of the amidated hemoglobin S increased the solubility of the protein further. The solubility of hemoglobin S with a double modification is nearly twice that of the unmodified protein and is close to that of 1:1 mixture of hemoglobin S and hemoglobin F. The results demonstrate the additivity of the solubilizing influence of perturbing the quinary interactions at the intermolecular contact regions of deoxy hemoglobin S.