Novel methods for the preparation and characterization of hyaluronan oligosaccharides of defined length
Novel methods for the preparation and characterization of hyaluronan oligosaccharides of defined length
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DOI：
10.1093/glycob/11.12.1025
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发表时间：
2001-12-01
期刊：
影响因子：
4.3
通讯作者：
Day, AJ
中科院分区：
文献类型：
--
作者：
Mahoney, DJ;Aplin, RT;Day, AJ
透明质酸是一种普遍存在的高分子量糖胺聚糖，作为细胞外基质的结构成分并介导细胞粘附。最近有许多报道称，透明质酸的片段与完整的分子相比具有不同的性质。虽然这些结果中的许多可能是真实的，但有些活性可能是由于所用制剂中的次要成分造成的。因此，在细胞生物学和结构研究中使用具有良好特征和高度纯化的低聚糖是很重要的，这样可以避免错误的结果。我们提出了用粒径排除法和阴离子交换色谱法在睾丸透明质酸酶消化透明质酸后纯化确定大小的透明质酸低聚物的方法。这些制剂通过电喷雾电离质谱、基质辅助激光解吸/电离质谱与飞行时间分析以及荧光团辅助碳水化合物电泳的组合进行了表征。透明质酸低聚物的范围从四糖到34米。4- 16米被证明是均匀的关于长度，但确实含有不同数量的硫酸软骨素。如果用医用级透明质酸而不是这里使用的相对不纯的起始材料进行消化，这种污染物就可以被最小化。18- 34-mer的制剂是不同长度的寡糖的混合物（例如，后者含有87%的34-mer， 10%的32-mer和3%的30-mer），但不含可检测的硫酸软骨素。除了具有偶数糖环的低聚物外，还鉴定了具有末端葡萄糖醛酸残基的新型5-和7-聚物。
Hyaluronan is a ubiquitous glycosaminoglycan of high molecular weight that acts as a structural component of extracellular matrices and mediates cell adhesion. There have been numerous recent reports that fragments of hyaluronan have different properties compared to the intact molecule. Though many of these results may be genuine, it is possible that some activities are due to minor components in the preparations used. Therefore, it is important that well-characterized and highly purified oligosaccharides are used in cell biological and structural studies so that erroneous results are avoided. We present methods for the purification of hyaluronan oligomers of defined size using size exclusion and anion-exchange chromatography following digestion of hyaluronan with testicular hyaluronidase. These preparations were characterized by a combination of electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight analysis, and fluorophore-assisted carbohydrate electrophoresis. Hyaluronan oligomers ranging from tetrasaccharides to 34-mers were separated. The 4- to 16-mers were shown to be homogeneous with regard to length but did contain varying amounts of chondroitin sulfate. This contaminant could have been minimized if digestion had been performed with medical-grade hyaluronan rather than the relatively impure starting material used here. The 18- to 34-mer preparations were mixtures of oligosaccharides of different lengths (e.g., the latter contained 87% 34-mer, 10% 32-mer, and 3% 30-mer) but were free of detectable chondroitin sulfate. In addition to oligomers with even numbers of sugar rings, novel 5- and 7-mers with terminal glucuronic acid residues were identified.