Peptides from the Site of Rabbit Antibody: Anti-3-Azopyridine Antibody

Abstract

The amino acid sequences of two peptides containing tyrosine that appears to be derived from the combining site of an anti-3-azopyridine antibody were determined. Binding studies showed that the antibody was inactivated by iodination and that the attack was in the site, since the presence of the hapten, pyridine, during iodination prevented the loss of sites. Peptides from the site were isolated after a paired labeling procedure as follows. One portion of the antibody was iodinated with ¹²⁵I-labeled hypoiodite. A second portion was iodinated with ¹³¹I-labeled hypoiodite in the presence of hapten to protect the site from iodination. The protected and unprotected preparations were mixed, digested with pepsin and the iodinated peptides separated. The ratio of ¹²⁵I/¹³¹I relative to that of the unfractionated digest was determined for the peptides; a high ratio was taken to indicate that the peptide was derived from the antibody site. Two high-ratio peptides, each in a yield of about 30% of the total antibody site initially present and probably from different populations of the anti-3P antibody, had the sequences Val-Ser-MIT (monoiodotyrosine) and MIT-Ser-Leu. The two peptides were shown to be derived from the heavy chain of the antibody and probably from the variable portion. A third high-ratio peptide, present in a much smaller amount (5% yield), was from the light chain fraction.