High-performance liquid chromatography of proteins on deformed non-porous agarose beads. Fast boronate affinity chromatography of haemoglobin at neutral pH.
High-performance liquid chromatography of proteins on deformed non-porous agarose beads. Fast boronate affinity chromatography of haemoglobin at neutral pH.
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DOI：
10.1016/s0021-9673(00)96091-6
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发表时间：
1990-02
期刊：
影响因子：
--
通讯作者：
S. Hjertén;J. Li
中科院分区：
文献类型：
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作者：
S. Hjertén;J. Li
将氨基苯硼酸连接到直径为12-15 μm的环氧活化的无孔琼脂糖珠上，并将该硼酸盐凝胶柱用于从非糖基化血红蛋白中分离糖基化血红蛋白。通过改变实验条件，表明第二峰（糖化血红蛋白）和第一峰（非糖化血红蛋白）之间的比值实际上与pH值（7-8）、离子强度、流速和上样量（0.7ml柱上至少160 μl溶血产物）无关。因此，没有必要为了获得可重复的结果而彻底控制这些参数，这在糖化血红蛋白的快速常规分析中是一个很大的优势。在4.0 ml/min的流速下，在2.5 cm X 0.6 cm I.D.上2 min内完成分析。柱分析的总时间也很短，因为来自一滴血的样品可以在溶血1分钟后直接上样到柱上，而不需要通过耗时的离心除去细胞碎片。实验在接近血红蛋白等电点的pH下进行，因为在该pH下血红蛋白具有可忽略的净电荷，因此不会与配体、基质和在先前运行中吸附的蛋白质的带电基团相互作用。
Aminophenylboronic acid was attached to epoxy-activated non-porous agarose beads with diameters of 12–15 μm and this boronate gel column was used for the fractionation of glycosylated from non-glycosylated haemoglobin. By varying the experimental conditions it was shown that the ratio between the second peak (glycosylated haemoglobin) and the first peak (non-glycosylated haemoglobin) was virtually independent of pH in the range 7–8, ionic strength, flow-rate and sample load (up to at least 160 μl ofhaemolysate on a 0.7-ml column). It is therefore not necessary to control thoroughly these parameters in order to obtain reproducible results, which is a great advantage in fast routine analyses of glycosylated haemoglobin. At a flow-rate of 4.0 ml/min an analysis was finished within 2 min on a 2.5 cm X 0.6 cm I.D. column. The total time of an analysis was also short because a sample from a droplet of blood could be applied directly onto the column after haemolysis for 1 min without removal of cell debris by time-consuming centrifugation. The experiments were performed at a pH close to the isoelectric point of haemoglobin, because at this pH haemoglobin has a negligible net charge and will therefore not interact with the charged groups of the ligands, the matrix and proteins adsorbed in previous runs.