NOVEL METHODS FOR ANALYZING GLYCOSMINOGLYCAN SUBSTRUCTURE

该项目的目的是开发新的分析方法， 高分辨率和高分辨率的糖胺聚糖亚结构 灵敏度 高性能阴离子交换器， Dionex（CarboPac）已经开发了稳定性和分辨能力 PA 1）用于分离糖和低聚糖。 当用于 适当的监测器（UV、荧光或脉冲安培法 检测器），可以实现ng范围内的检测极限。 糖胺聚糖可被酶选择性降解： 软骨素酶消化硫酸软骨素和透明质酸， 肝素酶消化肝素和硫酸乙酰肝素，以及角蛋白酶消化 硫酸角质素。 主要产物是单-、二-和三-的混合物。 三硫键，具有携带硫酸盐残基的各种位置。 我们 已经开发了一种还原方法，用于在 消化产物的还原末端。 这两个稳定他们， 碱并消除α和β端基异构体。 它还提供了一个 用于检测所有产物的高灵敏度荧光测定， 从而避免了其它方法的低检测限， 饱和（UV）或高度硫酸化（脉冲安培）产物。 的 用软骨素产生的二糖优化了方法 硫酸盐和透明质酸的软骨素酶，和二糖 由硫酸角质素和角质素酶产生。 当前主题 兴趣包括：（1）适应的方法来解决和纯化 不同长度的低聚糖， 透明质酸及其特异性裂解酶和消除酶 糖胺聚糖，（2）二糖的拆分和鉴定 由硫酸乙酰肝素与各种对此特异的酶产生 糖胺聚糖，和（3）该程序的应用，分析 硫酸软骨素和透明质酸的含量和组成， 骨关节炎患者滑液样品中的酸。

The purpose of this project is to develop new methods for analyzing the substructure of glycosaminoglycans with high resolution and high sensitivity. High performance anion exchangers with exceptional stability and resolving power have been developed by Dionex (CarboPac PA1) for separation of sugars and oligosaccharides. When used with appropriate monitors (UV, fluorescence, or pulsed amperometric detectors), detection limits in the ng range can be achieved. Glycosaminoglycans can be selectively degraded with enzymes: chondroitinases digest chondroitin sulfates and hyaluronic acid, heparinases digest heparin and heparan sulfate, and keratanases digest keratan sulfate. The major products are mixtures of mono-, di- and trisaccharides, with various positions carrying sulfate residues. We have developed a reductive method for introducing a fluorochrome on the reducing ends of the digestion products. This both stabilizes them to alkali and eliminates the alpha and beta anomers. It also provides a highly sensitive fluorescent assay for detecting all the products, thereby avoiding the low detection limits of the other procedures for saturated (UV) or highly sulfated (pulsed amperometric) products. The method has been optimized with disaccharides generated from chondroitin sulfate and hyaluronic acid by the chondroitinases, and disaccharides generated from keratan sulfate with keratanases. Topics of present interest include: (1) adaptation of the method to resolve and purify oligosaccharides with different lengths from partial digests of hyaluronic acid with lyase and eliminase enzymes specific for this glycosaminoglycan, (2) resolution and identification of the disaccharides generated from heparan sulfate with various enzymes specific for this glycosaminoglycan, and (3) the application of the procedure to analyze the contents and compositions of the chondroitin sulfate and hyaluronic acid in synovial fluid samples from patients with osteoarthritis.