The NH2-terminal region of the sickle hemoglobin beta chain. II. Characterization of monospecific antibodies.
We have previously shown that antibodies specific for hemoglobin S could be fractionated by absorption of an antiserum to hemoglobin S to Sepharose containing a synthetic oligopeptide. betaS (1-13), corresponding to the first 13 amino acid residues of the beta chain of hemoglobin S. We report here that this antibody population, anti-betaS (1-13), shows considerable restriction of heterogeneity in isoelectric focusing studies and monospecificity on velocity ultracentrifugation in the presence of hemoglobin S. The binding of various hemoglobin species to anti-betaS (1-13) was studied using a double antibody radioimmunoassay with [14C]carbamoylated hemoglobin S. Carbonmonoxy-, oxy-, met-, and cyanmethemoglobin S reacted equally with the antibody, but deoxyhemoglobin (with or without organic phosphates) reacted differently. Hemoglobin A and several hemoglobin mutants with alterations in the NH2-terminal region of the beta chain did not displace labeled hemoglobin S from anti-betaS (1-13). BETAS chains reacted with the antibody, but less well than hemoglobin S, while betaA and alpha chains, and globins did not react with the antibody. The synthetic peptide, betaS (1-13), used for fractionation, reacted with the antibody about 300-fold less efficiently than hemoglobin S. BetaS (3-13) was even less reactive, while smaller peptides which included the valine residue at position 6 displaced little of the tracer [14C]carbamoylated hemoglobin S at concentrations as high as 10(-2) M. We interpret these results to indicate that this method of immunoabsorption has produced a monospecific subfraction of antibodies which is specific for the NH2-terminal region of the beta chain of hemoglobin S in its native conformation.
D000595:Amino Acid Sequence; D000906:Antibodies; D001797:Blood Protein Electrophoresis; D002263:Carboxyhemoglobin; D006418:Heme / Q000032:analysis; D006451:Hemoglobin, Sickle; D006801:Humans; D007122:Immunoelectrophoresis; D007525:Isoelectric Focusing; D007700:Kinetics; D009842:Oligopeptides / Q000097:blood / Q000138:chemical synthesis; D011863:Radioimmunoassay
D000906:Antibodies; D006451:Hemoglobin, Sickle; D009842:Oligopeptides; D006418:Heme; D002263:Carboxyhemoglobin