Plaas A H, Hascall V C, Midura R J
Shriners Hospital for Crippled Children, Tampa Unit, FL 33612, USA.
Glycobiology. 1996 Dec;6(8):823-9. doi: 10.1093/glycob/6.8.823.
Sulfated glycosaminoglycans such as chondroitin sulfate are composed of three structural domains, a linkage oligosaccharide, connecting the chain to the core protein, a variably sulfated disaccharide repeat structure within the chain and a nonreducing terminal, and these domains may confer specific functions on particular chain populations. We report here a new and highly sensitive method for the detection and quantitation of all nonreducing terminal residues and internal disaccharides obtained by chondroitinase ABC or ACII digestion of aggrecan chondroitin sulfate. The procedure involves a quantitative reductive animation of the reducing ends of sulfated mono- and disaccharide chondroitinase products with 2-aminopyridine and boranedimethylamine. All derivatized saccharides can be separated and quantitated by fluorescence in a single chromatographic step on an AS4A anion exchange column, eluted with a gradient (0-500 nM) of sodium trifluoroacetate. The reproducibility and stability of the derivatisation, together with the sensitivity of the chromatography system, allowed for routine quantitation in the range of 3-500 pmol of reducing group (corresponding to about 1.5-250 ng of disaccharide or 0.75-125 ng of monosaccharide). Moreover, the fluorescence yield (fluorescence area units per pmol of reducing group) was virtually identical for all saccharides analyzed. Application of this method to an analysis of aggrecan purified from calf epiphyseal cartilage and from rat chondrosarcoma chondrocyte cultures allowed a precise identification and quantitation of the internal disaccharides and the nonreducing terminal structures, together with an estimation of the number average molecular weight of CS chains in these aggrecan preparations.
硫酸化糖胺聚糖，如硫酸软骨素，由三个结构域组成：一个连接寡糖，将链连接到核心蛋白；链内可变硫酸化的二糖重复结构；以及一个非还原末端，这些结构域可能赋予特定链群体特定功能。我们在此报告一种新的高灵敏度方法，用于检测和定量通过软骨素酶ABC或ACII消化聚集蛋白聚糖硫酸软骨素获得的所有非还原末端残基和内部二糖。该方法包括用2-氨基吡啶和硼烷二甲胺对硫酸化单糖和二糖软骨素酶产物的还原端进行定量还原胺化。所有衍生化的糖类可以在AS4A阴离子交换柱上通过荧光在单个色谱步骤中分离和定量，用三氟乙酸钠梯度（0-500 nM）洗脱。衍生化的重现性和稳定性，以及色谱系统的灵敏度，使得在3-500 pmol还原基团范围内（相当于约1.5-250 ng二糖或0.75-125 ng单糖）能够进行常规定量。此外，对于所有分析的糖类，荧光产率（每pmol还原基团的荧光面积单位）几乎相同。将该方法应用于从小牛骨骺软骨和大鼠软骨肉瘤软骨细胞培养物中纯化的聚集蛋白聚糖的分析，能够精确鉴定和定量内部二糖和非还原末端结构，同时估计这些聚集蛋白聚糖制剂中硫酸软骨素链的数均分子量。
