NOVEL METHODS FOR ANALYZING GLYCOSMINOGLYCAN SUBSTRUCTURE

该项目的目的是开发新的分析方法， 具有高分辨率和高分辨率的糖胺聚糖的亚结构 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或脉冲安培检测器），检测 可以实现ng范围内的限制。 糖胺聚糖可以是 选择性降解酶：软骨素酶降解软骨素 硫酸盐和透明质酸，肝素酶消化肝素和乙酰肝素 硫酸角质素和角质素酶消化硫酸角质素。 主要产品是 通常是单糖和二糖的混合物， 带有硫酸盐残留物的位置。 这些二糖不稳定 与用于净化阴离子交换柱的碱性条件有关。 我们 已经开发了一种硼氢化物还原方法， 二糖，既能使它们对碱稳定，又能消除" 和β端基异构体。 该方法已被优化与二糖 由细菌从硫酸软骨素和透明质酸产生 消除酶，软骨素酶ABC和软骨素酶AC。 当前主题 感兴趣的包括：（1）使用醋酸汞去除 来自消除酶消化反应的不饱和糖醛酸， 改变了除非还原末端以外的所有消化产物， 允许识别后者，（2）调整方法， 从部分寡糖中分离纯化不同长度的寡糖 透明质酸的裂解酶和消除酶特异性 （3）糖胺聚糖的分离和鉴定 由硫酸乙酰肝素与各种酶产生的二糖 特异于该糖胺聚糖和通过亚硝酸处理，和 (4)应用程序分析内容， 滑膜中硫酸软骨素和透明质酸的组合物 骨关节炎患者的体液样本。

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 or pulsed amperometric detectors), detection limits in the ng range can be achieved. Glycosaminoglycans can be selectively degraded with enzymes: chondroitinases digests chondroitin sulfates and hyaluronic acid, heparinases digest heparin and heparan sulfate, and keratanases digest keratan sulfate. The major product is often a mixture of monosaccharides and disaccharides, with various positions carrying a sulfate residue. These disaccharides are unstable to the alkali conditions used to elute the anion exchange column. We have developed a borohydride reduction method to reduce the disaccharides which both stabilizes them to alkali and eliminates the ` and beta anomers. The method has been optimized with disaccharides generated from chondroitin sulfate and hyaluronic acid by the bacterial eliminases, chondroitinase ABC and chondroitinase AC. Topics of present interest include: (1) the use of mercuric acetate to remove the unsaturated uronic acid from the eliminase digestion reaction which alters all the digestion products except the non-reducing ends and allows the latter to be identified, (2) 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, (3) resolution and identification of the disaccharides generated from heparan sulfate with various enzymes specific for this glycosaminoglycan and by nitrous acid treatments, and (4) 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.