Antigenic structure of human hemoglobin: Delineation of the antigenic site (site 2) within region 41-65 of the alpha chain by immunochemistry of synthetic peptides

A comprehensive synthetic approach consisting of a series of consecutive, uniform overlapping peptides encompassing the entire protein chain was recently used to determine the full antigenic profile of the α-chain of human hemoglobin (Hb). The peptides synthesized enabled the localization of five major "continuous" antigenic regions within the α chain. The present findings describe the delineation of an antigenic site (site 2) residing within the region 41-65. Ten peptides representing the α-chain regions 41-55, 51-65, 45-54, 45-56, 45-58, 45-60, 48-56, 49-56, 50-56, and 51-56 were synthesized and purified. Quantitative radioimmunoadsorbent titrations were used to determine binding to peptide adsorbents of radioiodinated anti-Hb antibodies that were raised in rabbit, goat, and outbred mouse. In one set of peptides, the N-terminal was fixed while the C-terminal end was increased by increments of two residues from Gln-54 to Lys-60 (i.e., peptides 45-54, 45-45, 45-58, and 45-60). Binding studies revealed that maximum antibody activity resided in peptide 45-45, indicating that Lys-56 marks the C-terminal boundary of the site. In the second set of peptides, the C-terminal was fixed at Lys-56 while the peptides were elongated at their N-terminal by one-residue increments from Gly-51 to Leu-48. Antibody-binding studies with these peptides indicated that Ser-49 defines the N-terminal boundary of the site. Therefore, the antigenic site within region 41-65 of the α chain comprises residues 49-56. The relevance of these findings to the immune recognition of Hb and other proteins is discussed.