Lin L C, Putnam F W
Proc Natl Acad Sci U S A. 1979 Dec;76(12):6572-6. doi: 10.1073/pnas.76.12.6572.
A myeloma IgD immunoglobulin (designated WAH) that was present in high concentration in plasma ( approximately 3.5 g/dl) was purified in >90% yield by a two-step procedure of ammonium sulfate precipitation plus AcA 34 gel filtration. Although the plasma had been stored for 2 years without the addition of a proteolytic inhibitor, no "spontaneous" degradation was apparent and the isolated IgD remained structurally intact. However, the purified IgD showed extreme susceptibility in vitro to various proteolytic enzymes; e.g., Fab(delta) (M(r) approximately 47,000) and Fc(delta) (M(r) approximately 80,000) fragments were generated quantitatively after only 10 min of incubation with papain in the absence of cysteine. By combining limited enzymatic digestion, reductive cleavage, and cyanogen bromide fragmentation, several series of well defined fragments corresponding to the different regions and domains of the IgD molecule were generated. These fragments are useful for physical, chemical, and immunological studies, as well as for the sequence determination of the IgD delta chain. A model of the IgD molecule was derived from such studies and from overlapping of the series of fragments. The possible existence of an extra constant domain in the delta chain appears unlikely in view of our finding of an extended hinge region of about 50 residues which can be cleaved off the amino terminus of the papain Fc(delta) by brief treatment with trypsin. In addition to a distinct stretch of carbohydrate attachment sites, the delta-chain hinge region contains a segment unusually rich in electrical charge. This charged segment is responsible for the lability of IgD to spontaneous degradation and may be related to its biological role as a B lymphocyte receptor.
一种在血浆中高浓度存在（约3.5 g/dl）的骨髓瘤IgD免疫球蛋白（命名为WAH），通过硫酸铵沉淀加AcA 34凝胶过滤的两步法进行纯化，产率超过90%。尽管血浆在未添加蛋白水解抑制剂的情况下储存了2年，但未观察到明显的“自发”降解，分离得到的IgD在结构上仍保持完整。然而，纯化后的IgD在体外对各种蛋白水解酶表现出极高的敏感性；例如，在没有半胱氨酸的情况下，与木瓜蛋白酶孵育仅10分钟后，就定量产生了Fab(delta)（分子量约47,000）和Fc(delta)（分子量约80,000）片段。通过结合有限的酶切、还原裂解和溴化氰裂解，产生了几系列对应于IgD分子不同区域和结构域的明确片段。这些片段可用于物理、化学和免疫学研究，以及IgD δ链的序列测定。IgD分子的模型是从这些研究以及片段系列的重叠推导出来的。鉴于我们发现了一个约50个残基的延长铰链区，用胰蛋白酶短暂处理可从木瓜蛋白酶Fc(delta)的氨基末端切除，因此δ链中额外恒定结构域的可能存在似乎不太可能。除了一段独特的碳水化合物附着位点外，δ链铰链区还包含一个电荷异常丰富的片段。这个带电荷的片段是IgD对自发降解不稳定的原因，可能与其作为B淋巴细胞受体的生物学作用有关。
