Poulsen K, Fraser K J, Haber E
Proc Natl Acad Sci U S A. 1972 Sep;69(9):2495-9. doi: 10.1073/pnas.69.9.2495.
The immunoglobulin light chain is thought to be organized into two structural domains, the constant and the variable regions, linked by a switch region of lesser tertiary organization. Light chains of homogeneous rabbit antibodies to pneumococcal polysaccharides labeled with (125)I were subjected to trypsin digestion at different temperatures. At 43 degrees molecular size (as determined by gel filtration) remained unchanged until the chain was subjected to full reduction and alkylation, when half molecules were demonstrated. One light chain resisted tryptic digestion at 43 degrees . At 50 degrees extensive digestion to small peptides occurred. These experiments confirm the susceptibility of the switch region to enzymatic attack as well as the relative resistance of the constant and variable region domains. They also demonstrate that the third disulfide bridge in rabbit light chain extends between the two structural domains and maintains the integrity of the molecule even when a peptide bond in the switch region is cleaved. When the light chain was subjected to dilute acid hydrolysis [10% acetic acid-pyridine (pH 2.5) in 7 M guanidine-HCl for 24-90 hr] selective cleavage at the switch region was demonstrated in yields up to 90%. The site of cleavage was shown to be the acid labile Asp 109-Pro 110 bond by automated Edman degradation. After cleavage the molecule remained intact until full reduction and alkylation, when half molecules were again demonstrated. A light chain cleaved in this way with its interdomain disulfide bond intact, was able to recombine with a homologous heavy chain and restore antigen binding. This indicates that an intact switch region is not needed for antibody activity. The availability of a functional variable region now allows for planning an examination of the antibody combining site by the technique of peptide synthesis.
免疫球蛋白轻链被认为由两个结构域组成，即恒定区和可变区，它们通过三级结构较弱的转换区相连。用（125）I标记的针对肺炎球菌多糖的兔同源抗体的轻链，在不同温度下进行胰蛋白酶消化。在43℃时，分子大小（通过凝胶过滤测定）保持不变，直到轻链完全还原和烷基化，此时出现半分子。一条轻链在43℃时抵抗胰蛋白酶消化。在50℃时，发生广泛消化形成小肽。这些实验证实了转换区对酶攻击的敏感性以及恒定区和可变区结构域的相对抗性。它们还表明，兔轻链中的第三个二硫键在两个结构域之间延伸，即使转换区的一个肽键被裂解，也能维持分子的完整性。当轻链进行稀酸水解[在7M盐酸胍中用10%乙酸-吡啶（pH2.5）处理24 - 90小时]时，在转换区显示出选择性裂解，产率高达90%。通过自动Edman降解表明裂解位点是酸不稳定的天冬氨酸109 - 脯氨酸110键。裂解后，分子保持完整，直到完全还原和烷基化，此时再次出现半分子。以这种方式裂解且其结构域间二硫键完整的轻链，能够与同源重链重组并恢复抗原结合。这表明抗体活性不需要完整的转换区。现在有了功能性可变区，就可以通过肽合成技术来计划对抗体结合位点的研究。
