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 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.