Effect of Reduction of Several Disulfide Bonds on the Properties and Recombination of Univalent Fragments of Rabbit Antibody
Effect of Reduction of Several Disulfide Bonds on the Properties and Recombination of Univalent Fragments of Rabbit Antibody
复制标题
多个二硫键还原对兔抗体单价片段性质和重组的影响
DOI：
--
复制
发表时间：
1963
影响因子：
4.8
通讯作者：
A. Nisonoff
中科院分区：
文献类型：
--
作者：
W. Mandy;A. Nisonoff
通过胃蛋白酶和还原剂同时或连续处理释放的兔抗体的单价片段在许多方面（l-3）与木瓜蛋白酶作用形成的单价片段相似(4)。相似之处包括平均分子量、沉降系数（3.5 S）、氨基酸组成、单位重量抑制特定沉淀的能力和色谱行为。在此基础上提出，胃蛋白酶和还原剂的作用机制与木瓜蛋白酶相似，与还原剂作为活化剂配合使用。分子中似乎有一个或多个区域特别容易受到任一种蛋白水解酶的作用。在这种攻击之后，导致去除一个大的无活性片段（波特分数III(4)），单价片段仅由一个单一的，不稳定的二硫键连接。Cebra等人(5)最近表明，与不溶性多肽共价连接的木瓜蛋白酶和还原剂可以进行两阶段反应。单独与胃蛋白酶反应产生5-S二价片段，分子量为106,000，还原后减少到56,000（1,2）。还原后的片段在超离心机中以单个3.5 s峰迁移的事实表明，还原后的5-S分子大约被分成两半。3.5-S碎片可以很容易地再氧化成5-S材料，收率很高。当使用特异性纯化的抗体时，形成沉淀的能力在再氧化后很大程度上恢复(3)。混合抗体可以通过将不同特异性的抗体片段的混合物再氧化而形成（6,7）。单价片段的重组似乎是随机的(7)。5-S向3.5-S蛋白的转化是由一个不稳定的二硫键的还原引起的(8)。用0.008 M 2-巯基乙胺盐酸盐在37℃和pH 5条件下处理1小时可使该键还原。在这些条件下，根据氨基酸分析，它是5-S分子中大约14个键中唯一断裂的二硫键(3)。研究额外的二硫键断裂后发生的聚集的性质是有意义的。如果其他的巯基变成
The univalent fragments of rabbit antibody released by simultaneous or successive treatments with pepsin and a reducing agent are similar in a number of respects (l-3) to those formed by the action of papain (4). The similarities include the average molecular weight, sedimentation coefficient (3.5 S), amino acid composition, capacity per unit weight to inhibit specific precipitation, and chromatographic behavior. It was proposed on this basis that the mechanism of action of pepsin and a reducing agent was similar to that of papain, which is used in conjunction with a reducing agent as activator. There appear to be one or more regions in the molecule that are particularly susceptible to the action of either proteolytic enzyme. Subsequent to this attack, which results in the removal of a large inactive fragment (Porter’s Fraction III (4)), the univalent fragments remain linked only by a single, labile disulfide bond. Cebra et al. (5) have shown recently that a two-stage reaction can be carried out with papain, covalently linked to an insoluble polypeptide, and a reducing agent. The reaction with pepsin alone yields a 5-S bivalent fragment with a molecular weight of 106,000, which is decreased to 56,000 upon reduction (1,2). The fact that the reduced fragments migrate as a single 3.5-S peak in the ultracentrifuge suggests that the 5-S molecule is split approximately in half on reduction. The 3.5-S fragments can readily be reoxidized in good yield to 5-S material. When specifically purified antibodies are used, the capacity to form precipitates is largely restored upon reoxidation (3). Hybrid antibodies can be formed by the reoxidation of a mixture of antibody fragments of different specificity (6, 7). The recombination of univalent fragments appears to bc random (7). The transformation of 5-S to 3.5-S protein is brought about by the reduction of one labile disulfidc bond (8). This bond can be reduced by treatment with 0.008 M 2-mercaptoethylamine hydrochloride for 1 hour at 37” and pH 5. Under these conditions, it is the only disulfide bond broken in the 5-S molecule, of approximately 14 bonds that appear to be present on the basis of amino acid analysis (3). It was of interest to study the nature of the aggregation that occurred after additional disulfide bonds were broken. If other sulfhydryl groups became available for