Location of light chain tyrosine peptides from the combining site of a rabbit anti-p-azobenzoate antibody.

Peptic peptides containing a tyrosyl residue from the binding site of a rabbit anti-p-azobenzoate antibody were isolated by means of the paired-iodination procedure. The peptides were from the light chain, and the tyrosyl residue is 29, 30, 31, 32, 32A, 32B, 33 at position 30 in the sequence -Val-Tyr-Asn-Asx-Lys-Gly-Leu- and thus is in the first hypervariable region. The sequence of the N-terminal 40 residues was determined. The major antibody-site peptide isolated was a diiodotyrosyl (DIT) tetrapeptide representing residues 30-32A; the monoiodotyrosyl (MIT) tetrapeptide was also isolated, but in a smaller yield. By isoelectric focusing, the light chain appeared to be homogeneous. No heterogeneity was apparent in the light chain sequencing until position 32B when, in addition to the phenylthiohydantoin derivative of tyrosine present as the major residue, a significant amount of the phenylthiohydantoin derivative of glycine was obtained. The glycine presumably represents a light chain variant population and explains the source of the other antibody-site peptides isolated, i.e. two pentapeptides, apparently of the sequence Tyr-Asn-Asx-Lys-Gly, isolated as the DIT and MIT derivatives. The tetrapeptides must have been derived from the peptic cleavage between Lys 32A and Tyr 32B in the major light chain variant and the pentapeptides from the peptic cleavage between Gly 32B and Leu 33 in the other variant. It is interesting that position 30 is occupied by a tyrosyl residue in five out of twelve other rabbit antibody light chains of known sequence (Margolies, M.N. et al., Proc. Nat. Acad. Sci. US 1975.72: 2180). One light chain is from another rabbit anti-p-azobenzoate antibody in which Tyr 30 is apparently not important in hapten binding although a tyrosyl at position 96 is clearly involved in hapten binding (Roholt, O.A. et al., J. Immunol. 1973.111:1367). The other four of the five light chains are from anti-pneumococcal polysaccharide antibodies in which the role of this tyrosyl residue is not known.