Hemoglobin binding with haptoglobin: delineation of the haptoglobin binding site on the alpha-chain of human hemoglobin.

Previous studies from this laboratory employing a comprehensive synthetic overlapping peptide strategy showed that the alpha-chain of human hemoglobin (Hb) contains a single haptoglobin (HP) binding region residing within residues alpha 121-135. The present study describes a precise delineation of this Hp-binding site on the alpha-chain. Two overlapping peptides (alpha 111-125 and alpha 121-135) spanning this region and a panel of five peptides decreasing at the C-terminal from residue 135 by decrements of two residues (alpha 119-135, alpha 119-133, alpha 119-131, alpha 119-129, and alpha 119-127) were synthesized, purified, and characterized. Quantitative radiometric titration of 125I-labeled human HP (type 2-1) with adsorbents of each of these synthetic peptides showed that the peptide alpha 119-127 retained a Hp-binding activity equivalent to that of peptide alpha 121-135. 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 (alpha 122-127, alpha 121-127, alpha 120-127, alpha 119-127, alpha 118-127, alpha 117-127, alpha 116-127, and alpha 115-127). The binding of 125I-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 alpha-chain of human Hb comprises residues alpha 121-127.