Seldin D C, Seno N, Austen K F, Stevens R L
Anal Biochem. 1984 Aug 15;141(1):291-300. doi: 10.1016/0003-2697(84)90459-7.
A high-performance liquid chromatography method for analyzing disaccharides derived from chondroitin sulfate glycosaminoglycans has been developed which employs a Whatman Partisil-10 PAC amino-cyano column and an acetonitrile/methanol/ammonium acetate solvent to resolve disulfated, monosulfated, and unsulfated disaccharides in a chromatographic run of less than 20 min. The single known trisulfated chrondroitin disaccharide can be eluted in an alternate solvent system containing the same mobile phase components in different proportions. Disaccharides were prepared for chromatography from glycosaminoglycans and proteoglycans of known compositions by digestion with chondroitinase ABC, with the exception of king crab cartilage glycosaminoglycan which was incubated sequentially with hyaluronidase and chondroitinase ABC. Disaccharides were extracted from the digestion mixtures in 80% ethanol, dried over nitrogen, resuspended in the HPLC solvent, and chromatographed at a flow rate of 1 ml/min. Unsaturated disaccharides in the column eluate were detected by continuous ultraviolet absorbance monitoring at 232 nm; alternatively, fractions were collected and assayed for uronic acid content or radioactivity. By utilizing the HPLC technique in conjunction with chondroitinase ABC and AC digestion and sulfatase hydrolysis, the epimeric structures of chondroitin sulfates E and H were confirmed. With this technique, rapid and reproducible analyses of chondroitin sulfate disaccharides generated from mouse mast cell proteoglycan and from glycosaminoglycans of squid cranial cartilage, shark skin, hagfish skin, and hagfish notocord were in close agreement with compositions obtained by other techniques.
已开发出一种用于分析硫酸软骨素糖胺聚糖衍生二糖的高效液相色谱方法，该方法采用沃特曼Partisil - 10 PAC氨基氰基柱和乙腈/甲醇/醋酸铵溶剂，可在不到20分钟的色谱运行中分离双硫酸化、单硫酸化和未硫酸化的二糖。单一已知的三硫酸化软骨素二糖可在含有相同流动相成分但比例不同的替代溶剂系统中洗脱。通过用软骨素酶ABC消化，从已知组成的糖胺聚糖和蛋白聚糖中制备用于色谱分析的二糖，但帝王蟹软骨糖胺聚糖除外，它先与透明质酸酶孵育，然后再与软骨素酶ABC孵育。二糖从消化混合物中用80%乙醇萃取，经氮气吹干，重悬于高效液相色谱溶剂中，并以1 ml/min的流速进行色谱分析。通过在232 nm处连续监测紫外吸光度来检测柱洗脱液中的不饱和二糖；或者，收集馏分并测定糖醛酸含量或放射性。通过将高效液相色谱技术与软骨素酶ABC和AC消化以及硫酸酯酶水解相结合，证实了硫酸软骨素E和H的差向异构体结构。利用该技术，对从小鼠肥大细胞蛋白聚糖以及鱿鱼颅软骨、鲨鱼皮、盲鳗皮和盲鳗脊索的糖胺聚糖产生的硫酸软骨素二糖进行快速且可重复的分析，结果与通过其他技术获得的组成非常一致。
