Practical Application of Fluorescence Labeling to Component Sugar Analysis of Glycoproteins

报道了一种用pyridylamination法测定糖蛋白组分糖的方法（H. Takemoto, et al. 1985）。该方法包括糖蛋白的酸水解，氨基的n -乙酰化，用NaBH_3CN， 2-氨基吡啶和HCl对单糖的还原端进行吡啶层化，用凝胶色谱（HPLC）纯化糖的吡啶氨基（PA-）衍生物，用反相HPLC分析PA-糖。为研制全自动糖分析仪，改进了吡啶层合工艺条件。1)挥发性试剂，如二甲胺。用硼烷络合物和2-氨基吡啶-乙酸代替NaBH_3CN和2-氨基吡啶-盐酸。2)选择反应条件获得pa -糖的最大产率（中性糖70%，n -乙酰氨基糖87%）。3)开发了减压氮气流，使试剂有效蒸发。利用新的反应条件和蒸发过程，可以省去凝胶色谱法（HPLC）纯化pa -糖。采用自动进样器和数据处理机对合成的pa糖进行高效液相色谱分析。糖蛋白酸水解产物的糖用量为1 ~ 10 nmol，得到了合理的结果。

A method for determination of component sugars of glycoproteins by pyridylamination was reported (H. Takemoto, et al. 1985). The method includes acid hydrolysis of glycoprotein, N-acetylation of amino groups, pyridylamination of the reducing ends of the monosaccharides with NaBH_3CN, 2-aminopyridine, and HCl, purification of pyridylamino (PA-) derivatives of sugars by gel chromatography (HPLC), and the analysis of PA-sugars by reversed-phase HPLC. The pyridylamination conditions were improved for the purpose of the making of an automatic sugar analyzer. 1) Volatile reagents such as dimethylamine. borane complex, and 2-aminopyridine-acetic acid were used intead of NaBH_3CN and 2-aminopyridine-HCl. 2) The reaction conditions were chosen to obtain maximum yield of PA-sugars (neutral sugars 70% and N-acetylaminosugars 87%). 3) Nitrogen flow under reduced pressure was developed to evaporate the reagents effectively. Using the new reaction conditions and the evaporation procedre, purification of PA-sugars by gel chromatography (HPLC) could be omitted. An automatic pyridylamination apparatus using the new conditions was constructed and PA-sugars thus obtained were analyzed by HPLC equipped with an automatic injector and a data processor. Reasonable results were obtained by using 1-10 nmol of sugars of acid hydrolysates of glycoproteins.