Guidelines for the preparative fractionation of human serum proteins on gradient-eluted columns of concanavalin A-sepharose: elution positions of fourteen well-characterized proteins and evidence for concanavalin A-reactive albumin-IgA and -IgG complexes.
Guidelines for the preparative fractionation of human serum proteins on gradient-eluted columns of concanavalin A-sepharose: elution positions of fourteen well-characterized proteins and evidence for concanavalin A-reactive albumin-IgA and -IgG complexes.
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伴刀豆球蛋白 A-琼脂糖梯度洗脱柱上人血清蛋白的制备级分离指南：14 个已充分表征的蛋白质的洗脱位置以及伴刀豆球蛋白 A 反应性白蛋白-IgA 和 -IgG 复合物的证据。
DOI：
10.1080/00327488308068176
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
J. Baumstark
中科院分区：
文献类型：
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作者：
J. Baumstark
已经对伴刀豆球蛋白 A-琼脂糖梯度洗脱柱上的血清蛋白分级进行了系统研究，以确定寡糖残基是否有足够的差异以允许合理的分离，并确定这些蛋白质将在色谱图中的哪个位置被洗脱。将人全血清和由其衍生的硫酸铵级分与2.1×75cm的伴刀豆球蛋白A-琼脂糖柱和4×400ml梯度(Varigard)结合使用，其中0.5M甲基α-D-吡喃葡萄糖苷作为极限缓冲液。 14 种充分表征的人血清蛋白的洗脱位置和色谱限已通过在琼脂糖凝胶中针对特定抗体的流出物级分等分试样（10X 浓缩）的双重扩散来确定，并通过免疫电泳确定了蛋白质的一般色谱分布。总体而言，结果表明寡糖侧链的组成（如分子大小、溶解度和电荷密度的差异）是从血清中色谱分离蛋白质的有用参数。尽管众所周知白蛋白是一种非糖蛋白，但 1.0% 的蛋白质与刀豆球蛋白 A-Sepharose 紧密结合。随后的实验表明，白蛋白结合是由于与 IgA 和 IgG 形成复合物所致，这两种蛋白都具有强结合所需的伴刀豆球蛋白 A 反应残基的补充。 2-巯基乙醇还原的白蛋白-IgA和-IgG复合物的十二烷基硫酸钠聚丙烯酰胺凝胶电泳产生与白蛋白的分子量以及IgA和IgG的重链和轻链相对应的条带，而未还原的样品没有解离。当这些复合物与刀豆球蛋白 A-Sepharose 反应并用 2-巯基乙醇处理时，游离白蛋白被洗脱。剩余的吸附糖蛋白、IgA 和 IgG 可用甲基 α-D-吡喃葡萄糖苷洗脱。这些结果强烈表明这些蛋白质和白蛋白通过二硫键连接。
Systematic studies on the fractionation of serum proteins on gradient-eluted columns of concanavalin A-Sepharose have been carried out to determine if the oligosaccharide residues were sufficiently different to permit a reasonable separation and to determine where in the chromatogram these proteins would be eluted. Human whole serum and ammonium sulfate fractions derived therefrom were used in conjunction with 2.1 x 75 cm columns of concanavalin A-Sepharose and a 4 x 400 ml gradient (Varigard) with 0.5 M methyl alpha-D-glucopyranoside as limit buffer. The elution positions and chromatographic limits of 14 well-characterized human serum proteins have been determined by double diffusion of aliquots of the effluent fractions (10X concentrated) in agarose gel against specific antibody and the general chromatographic distribution of the proteins by immunoelectrophoresis. Overall, the results demonstrate that the composition of the oligosaccharide side chain, like differences in molecular size, solubility, and charge density, is a useful parameter in the chromatographic separation of protein from serum. Although it is well-known that albumin is a nonglycoprotein, 1.0% of the protein was tightly bound by concanavalin A-Sepharose. Subsequent experiments showed that albumin binding was due to complex formation with IgA and IgG both of which possess the necessary complement of concanavalin A-reactive residues for strong binding. Sodium dodecylsulfate polyacrylamide gel electrophoresis of 2-mercaptoethanol-reduced albumin-IgA and -IgG complexes produced bands corresponding to the molecular weights of albumin and the heavy and light chains of IgA and IgG whereas unreduced samples were not dissociated. When these complexes were reacted with concanavalin A-Sepharose and treated with 2-mercaptoethanol, free albumin was eluted. The remaining adsorbed glycoprotein(s), IgA and IgG, could be eluted with methyl alpha-D-glucopyranoside. These results strongly suggest that these proteins and albumin are linked via a disulfide bond(s).