AFFINITY-LABELED RESIDUES IN ANTIBODY ACTIVE SITES .2. NEAREST-NEIGHBOR ANALYSES

By the method of affinity labeling, tyrosine residues in the active sites of a number of different antihapten IgG antibodies have been specifically modified. These residues occur on both heavy and light chains of the antibodies. In order to characterize these residues, small peptide fragments bearing the label have been prepared by Nagarse digestion, and have been isolated in a pure state in high yields, from heavy and light chains of rabbit and mouse anti-2,4-dinitrophenyl antibodies that were affinity labeled with [3H]m-nitrobenzenediazonium fiuoroborate. The isolated labeled peptides were shown to be dipeptides with m-nitrobenzeneazotyrosine as the carboxyl-terminal residue, and with a different characteristic amino acid as the predominant amino-terminal residue from each of three of the chain preparations. In the case of the light chains from the mouse antibodies, the predominant labeled dipeptide was aspartyl-(or asparginyl-)m-nitrobenzeneazotyrosine. By comparison with published sequences of mouse x-type Bence-Jones proteins, this result establishes that the labeled tyrosine residue is not in the invariant half of the light chains. This proves that the variable half of the light chain participates in the antibody active site. The results are consistent with the suggestion that the labeled tyrosine residues on heavy and light chains are homologous; that is, that they occur at comparable positions in the amino acid sequences of both types of chains. This suggestion leads to the postulate that a light chain and the Fd piece of a heavy chain are related to one another by a dyad axis of pseudosymmetry in each half of an intact immunoglobin G molecule. © 1969, American Chemical Society. All rights reserved.