Hemoglobin binding with haptoglobin: Delineation of the haptoglobin binding site on the α-chain of human hemoglobin

Previous studies from this laboratory employing a comprehensive synthetic overlapping peptide strategy showed that the α-chain of human hemoglobin (Hb) contains a single haptoglobin (HP) binding region residing within residues α121–135. The present study describes a precise delineation of this Hp-binding site on the α-chain. Two overlapping peptides (α111–125 and α121–135) spanning this region and a panel of five peptides decreasing at the C-terminal from residue 135 by decrements of two residues (α119–135, α119–133, α119–131, α119–129, and α119–127) were synthesized, purified, and characterized. Quantitative radiometric titration of125I-labeled human HP (type 2-1) with adsorbents of each of these synthetic peptides showed that the peptide α119–127 retained a Hp-binding activity equivalent to that of peptide α121–135. This finding indicated that Lys-127 marked the C-terminal boundary of the binding site. Another panel of eight peptides was then synthesized, which had their C-terminus fixed at Lys-127 and increased at the N-terminus by one-residue increments from residue 122 up to residue 115 (α122–127, α121–127, α120–127, α119–127, α118–127, α117–127, α116–127, and α115–127). The binding of125I-Hp to adsorbents of these peptides demonstrated that the N-terminal boundary of the site did not extend beyond Valine 121. It is, therefore, concluded that the Hp-binding site on the α-chain of human Hb comprises residues α121–127.