Conformation-specific antibodies to the α chain COOH terminus of hemoglobin A₀

An anti-hemoglobin antiserum obtained from a sheep immunized with human carboxyhemoglobin A₀ demonstrated little difference in its reactivity with deoxy- or carboxyhemoglobin A₀. However, a subpopulation of this antiserum isolated by synthetic peptide affinity chromatography clearly distinguished between these two hemoglobin species. This subpopulation, designated α(129-141) anti-hemoglobin antibodies, represents less than 1% of the total anti-hemoglobin antibodies. They are nonprecipitating by Ouchterlony analysis, and fluorescence-quenching studies demonstrate the interaction of a single antibody binding site per hemoglobin dimer. These antibodies bind preferentially to carboxyhemoglobin with a median affinity constant of 5 x 10⁸ M^-1 compared to binding to deoxyhemoglobin with a binding affinity of less than 1 x 10⁸ M^-1. Furthermore, the presence of these antibodies in stoichiometric amounts increases the oxygen affinity of hemoglobin, and thus antibody and oxygen binding to hemoglobin can be considered as a linked function.