Practical Application of Fluorescence Labeling to Component Sugar Analysis of Glycoproteins

荧光标记在糖蛋白成分糖分析中的实际应用

• 批准号: 62880019
• 负责人: HASE Sumihiro
• 金额: $ 8.77万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research
• 财政年份: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1988
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-62880019/
• 关键词: Fluorescence labeling; Component sugar analysis; ピリジルアミノ化

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.

本文报道了一种用吡啶胺化法测定糖蛋白组分糖的方法。Takemoto等人，1985）。该方法包括糖蛋白的酸水解、氨基的N-乙酰化、单糖还原端用NaBH_3CN、2-氨基吡啶和HCl的吡啶胺化、糖的吡啶氨基（PA-）衍生物的凝胶色谱（HPLC）纯化和PA-糖的反相HPLC分析。为了制作自动糖分析仪，对吡啶胺化条件进行了改进。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-HCl。2)选择反应条件以获得PA-糖的最大产率（中性糖70%和N-乙酰氨基糖87%）。3)开发了减压下的氮气流以有效地蒸发试剂。使用新的反应条件和蒸发过程，可以省略通过凝胶色谱法（HPLC）纯化PA-糖。构建了使用新条件的自动吡啶胺化装置，并通过配备有自动进样器和数据处理器的HPLC分析由此获得的PA-糖。通过使用1-10 nmol糖蛋白酸水解产物的糖获得合理的结果。

项目成果

S. Hase: "Analysis of Tissue Glycoprotein Sugar Chains by Two-Dimensional High-Performance Liquid Chromatographic Mapping" J. Chromatogr.434. 51-60 (1988)

S. Hase：“通过二维高效液相色谱图分析组织糖蛋白糖链”J. Chromatogr.434。

S.Hase: Anal.Biochem.67. 321-326 (1987)

S.Hase：Anal.Biochem.67。

S. Hase: "A New Trisaccharide Sugar Chain Linked to a Serine Residue in Bovine Blood Coagulation Factors VII and IX" J. Biochem.104. 867-868 (1988)

S. Hase：“与牛凝血因子 VII 和 IX 中的丝氨酸残基相连的新三糖糖链”J. Biochem.104。

S.Hase: J.Biochem.104. 867-868 (1988)

S.Hase：J.Biochem.104。

S.Yamamoto: J.Biochem.105. inpress (1989)

S.Yamamoto：J.Biochem.105。