Practical Application of Fluorescence Labeling to Component Sugar Analysis of Glycoproteins

报道了一种用吡啶层析法测定糖蛋白组成糖的方法(H.Takemoto等人)。1985年)。该方法包括糖蛋白的酸解、氨基的N-乙酰化、单糖还原端用NaBH_3CN、2-氨基吡啶和HCl的吡啶层析、用凝胶色谱(HPLC)纯化糖的吡啶氨基(PA-)衍生物，以及用反相高效液相色谱法分析PA-糖。为研制全自动糖分析仪，对吡啶层析条件进行了改进。1)二甲胺等挥发性试剂。在NaBH_3CN和2-氨基吡啶-盐酸盐之间使用了硼烷络合物和2-氨基吡啶-冰醋酸。2)对反应条件进行了选择，以获得最高的PA-糖得率(中性糖70%，N-乙酰氨基糖87%)。3)为了有效地蒸发试剂，开发了减压氮气流动。采用新的反应条件和蒸发过程，可以省去凝胶层析(HPLC)纯化PA-糖的步骤。在此基础上，建立了一套新条件下的自动吡啶层析装置，并用配有自动进样器和数据处理机的高效液相色谱仪对得到的PA-糖进行了分析。糖蛋白酸性水解物中糖的用量为1-10nmol时，结果较为合理。

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.