Baumstark J S
Prep Biochem. 1983;13(4):315-45. doi: 10.1080/00327488308068176.
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).
已对伴刀豆球蛋白A - 琼脂糖梯度洗脱柱上血清蛋白的分级分离进行了系统研究，以确定寡糖残基是否存在足够差异，从而实现合理分离，并确定这些蛋白质在色谱图中的洗脱位置。人全血清及其衍生的硫酸铵分级组分与2.1×75 cm的伴刀豆球蛋白A - 琼脂糖柱以及以0.5 Mα - D - 甲基吡喃葡萄糖苷为极限缓冲液的4×400 ml梯度（Varigard）一起使用。通过将流出级分（10倍浓缩）的等分试样在琼脂糖凝胶中与特异性抗体进行双向扩散，确定了14种特征明确的人血清蛋白的洗脱位置和色谱极限，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，寡糖侧链的组成与分子大小、溶解度和电荷密度的差异一样，是血清中蛋白质色谱分离的一个有用参数。虽然众所周知白蛋白是非糖蛋白，但1.0%的该蛋白与伴刀豆球蛋白A - 琼脂糖紧密结合。后续实验表明，白蛋白的结合是由于与IgA和IgG形成复合物，二者都具有与伴刀豆球蛋白A反应性残基结合所需的互补成分。对经2 - 巯基乙醇还原的白蛋白 - IgA和 - IgG复合物进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳，产生了与白蛋白以及IgA和IgG的重链和轻链分子量相对应的条带，而未还原的样品未解离。当这些复合物与伴刀豆球蛋白A - 琼脂糖反应并用2 - 巯基乙醇处理时，游离白蛋白被洗脱。其余吸附的糖蛋白（IgA和IgG）可用α - D - 甲基吡喃葡萄糖苷洗脱。这些结果强烈表明这些蛋白质与白蛋白是通过二硫键连接的。
