Specific modification of the carboxyl groups of hemoglobin S

The reactivity of the carboxyl groups of hemoglobin S to form amide bonds with glycine ethyl ester by carbodiimide-activated coupling, and the influence of this derivatization on the functional properties of the protein have been investigated. Incubation of carbonmonoxy or oxyhemoglobin S with 20 mM 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide in the presence of 100 mM [¹⁴C]glycine ethyl ester, at pH 6.0 and 23° C for 1 h resulted in the modification of, on an average three carboxyl groups of the protein. The Hill coefficient of the modified hemoglobin S was 2.7. indicating normal subunit interactions. The derivatization increased the oxygen affinity of the molecule (the p₅₀ was lowered from 8.0 to 5.0). The derivatization also resulted in an increase in the minimum gelling concentration of hemoglobin S from 16 to 24 g/100 ml. The reaction conditions used for the derivatization of the carboxyl groups of hemoglobin S are very selective for the protein carboxyl groups; very little of the label is associated with the heme carboxyls. Tryptic peptide mapping of the modified hemoglobin S indicated that the peptide β T₅, i.e. the segment representing amino acid residues 41 to 59 of β-chain, accounted for nearly 75% of the label associated with globin, demonstrating the high selectivity of the derivatization. Sequence analysis of the derivatized β T₅ demonstrated that at least 65% of the label incorporated into hemoglobin S is targeted toward the carboxyl groups of Glu-43(β), identifying it as the most reactive carboxyl group of hemoglobins. The results suggest that modification of the carboxyl group of hemoglobin S, presumably the γ-carboxyl of Glu-43(β), reduces the propensity of deoxyhemoglobin S to polymerize.