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.