Combined lectin-affinity and metal-interaction chromatography for the separation of glycophorins by high-performance liquid chromatography.
Combined lectin-affinity and metal-interaction chromatography for the separation of glycophorins by high-performance liquid chromatography.
复制标题
结合凝集素亲和和金属相互作用色谱，通过高效液相色谱分离血型糖蛋白。
DOI：
10.1016/s0021-9673(00)90549-1
复制
发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Horne,W
中科院分区：
文献类型：
--
作者：
Corradini,D;elRassi,Z;Horváth,C;Guerra,G;Horne,W
在高效液相色谱柱上采用凝集素亲和层析和金属相互作用层析对人红细胞唾液糖蛋白进行了层析。用固定在微颗粒二氧化硅载体上的含有小麦胚芽凝集素的柱将糖蛋白A、B和C与其他蛋白和糖蛋白E分离。以0.25 mg氯化钠为流动相，将糖蛋白吸附在凝集素柱上，用0.2 mg -乙酰氨基葡萄糖溶液逐步解吸回收。糖蛋白A、B和C在铜（II）螯合形式的二氧化硅结合亚氨基二乙酸固定相上分离成单独的组分。通过降低盐梯度洗脱，实现了金属相互作用层析对糖蛋白的分离。单个糖蛋白的保留时间和分辨率对初始氯化钠浓度和洗脱液的pH值敏感。在洗脱液中加入甲醇提高了分辨率。考察了25 mMphosphate缓冲液中pH和甲醇的线性梯度和递减梯度对糖蛋白分离的影响。在两种色谱中，流动相均含有0.05% （w/v）十二烷基硫酸钠。在辛基糖苷或CHAPS洗脱液中不能洗脱糖蛋白A和C。采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳法对所有色谱结果进行分析。
Human erythrocyte sialoglycoproteins, or glycophorins, were chromatographed by lectin-affinity and metal-interaction chromatography on high-performance liquid chromatographic columns. Glycophorins A, B and C were separated from other proteins and from glycophorin E by using a column containing wheat germ agglutinin, immobilized on a microparticulate silica support. The glycophorins were adsorbed on the lectin column from a mobile phase containing 0.25Msodium chloride and recovered by stepwise desorption with 0.2MN-acetylglucosamine solution. Glycophorins A, B and C were separated into the individual components on a silica-bound iminodiacetic acid stationary phase in the copper(II) chelate form. The separation of the glycophorins by metal-interaction chromatography was accomplished by decreasing salt gradient elution. Retention times and resolution of the individual glycophorins were sensitive to the initial sodium chloride concentration and the pH of the eluent. Addition of methanol to the eluent increased the resolution. The effects of linear, decreasing gradients of pH and methanol in 25 mMphosphate buffer on the resolution of glycophorins were also investigated. In both types of chromatography the mobile phases contained 0.05% (w/v) sodium dodecyl sulfate. With octylglycoside or CHAPS in the eluent glycophorins A and C could not be eluted. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze all the chromatographic results.