Friedenson B, Roholt O A, Appella E, Wright J D, Tissot R, Cohen C, Pressman D
Eur J Immunol. 1976 May;6(5):321-6. doi: 10.1002/eji.1830060504.
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.
通过配对碘化法分离出了含有来自兔抗对氨基苯甲酸抗体结合位点的酪氨酰残基的消化肽。这些肽来自轻链，酪氨酰残基在序列-Val-Tyr-Asn-Asx-Lys-Gly-Leu-的第30位，分别为29、30、31、32、32A、32B、33位，因此位于第一个高变区。测定了N端40个残基的序列。分离出的主要抗体位点肽是代表30-32A残基的二碘酪氨酰（DIT）四肽；单碘酪氨酰（MIT）四肽也被分离出来了，但产量较低。通过等电聚焦，轻链似乎是均一的。在轻链测序中，直到32B位之前都没有明显的异质性，在32B位，除了作为主要残基存在的酪氨酸的苯硫代乙内酰脲衍生物外，还获得了大量甘氨酸的苯硫代乙内酰脲衍生物。甘氨酸大概代表了一个轻链变异群体，并解释了分离出的其他抗体位点肽的来源，即两种五肽，显然其序列为Tyr-Asn-Asx-Lys-Gly，以DIT和MIT衍生物形式被分离出来。这些四肽一定是来自主要轻链变异体中32A位赖氨酸和32B位酪氨酸之间的消化裂解，而五肽则来自另一种变异体中32B位甘氨酸和33位亮氨酸之间的消化裂解。有趣的是，在已知序列的其他12种兔抗体轻链中，有5种轻链的第30位被酪氨酰残基占据（Margolies, M.N.等人，《美国国家科学院院刊》1975年。72: 2180）。一种轻链来自另一种兔抗对氨基苯甲酸抗体，其中30位的酪氨酸在半抗原结合中显然不重要，尽管96位的酪氨酰残基显然参与了半抗原结合（Roholt, O.A.等人，《免疫学杂志》1973年。111:1367）。这5种轻链中的另外4种来自抗肺炎球菌多糖抗体，其中这个酪氨酰残基的作用尚不清楚。
