Takagaki K, Takeda Y, Nakamura T, Daidouji K, Narita H, Endo M
Department of Biochemistry, Hirosaki University School of Medicine, Japan.
J Biochem Biophys Methods. 1994 Jun;28(4):313-20. doi: 10.1016/0165-022x(94)90007-8.
Glycosaminoglycan chains were liberated from proteoglycan by successive digestion with protease and endo-beta-xylosidase. The glycosaminoglycan chains were then labeled with a fluorescent reagent, 2-aminopyridine, by reductive amination. The resulting pyridylamino-glycosaminoglycans, including hyaluronic acid, heparan sulfate, chondroitin sulfate/dermatan sulfate and heparin, were separated by ion-exchange high-performance liquid chromatography using a TSK gel SAX analytical column with a limit of sensitivity in the picomol range. With the combined use of a dermatan sulfate-degrading enzyme, chondroitinase B, chondroitin sulfate and dermatan sulfate were identified and quantified, separately. About 50 mg of wet animal tissue was enough for analysis of each glycosaminoglycan with satisfactory results. This method facilitates rapid separation and microanalysis of glycosaminoglycans.
通过用蛋白酶和内切β-木糖苷酶连续消化，从蛋白聚糖中释放出糖胺聚糖链。然后通过还原胺化用荧光试剂2-氨基吡啶标记糖胺聚糖链。使用TSK凝胶SAX分析柱，通过离子交换高效液相色谱法分离所得的吡啶基氨基糖胺聚糖，包括透明质酸、硫酸乙酰肝素、硫酸软骨素/硫酸皮肤素和肝素，检测灵敏度极限在皮摩尔范围内。联合使用硫酸皮肤素降解酶、软骨素酶B，可分别鉴定和定量硫酸软骨素和硫酸皮肤素。约50mg湿动物组织足以对每种糖胺聚糖进行分析并获得满意结果。该方法有助于糖胺聚糖的快速分离和微量分析。
