NOVEL METHODS FOR ANALYZING GLYCOSMINOGLYCAN SUBSTRUCTURE

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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.