Preparation and Circular Dichroism Analysis of Sodium Hyaluronate Oligosaccharides and Chondroitin

Sodium hyaluronate was cleaved into an homologous series of oligosaccharides by the action of bovine testicular hyaluronidase (EC 3.2.1.35), an endo-β-hexosaminidase. Digestion conditions and gel-filtration chromatographic fractionation were optimized to produce oligosaccharides, clearly separable into peaks corresponding to 1-23 disaccharide units of the type D-glucuronosyl→N-acetyl-oglucosamine. The chromatographic method was also employed for the purification of a second homologous group of oligosaccharides, with the reversed sequence of monosaccharide units, produced by the action of leech hyaluronidase (EC 3.2.1.36), an endo-β-glucuronidase. Circular dichroism (CD) analysis (in the 200-250-nm range) of the oligosaccharides showed that the CD spectrum of hyaluronate in aqueous solution at neutral pH does not reflect to any substantial degree a polymer conformation which requires cooperative interaction between several repeating residues for stabilization. The enhanced CD properties of hyaluronate relative to those of monosaccharides are primarily related to the existence of the β-1,4 linkage from N-acetyl-D-glucosamine to D-glucuronate. Chondroitin, the N-acetyl-D-galactosamine analogue of hyaluronate, was prepared by chemical desulfation of chondroitin 4- and 6-sulfates. The purified product had a molecular weight range of 4000-8000 (10-20 disaccharide units). It was digested with testicular hyaluronidase, and the split products were isolated by gel filtration. In contrast to hyaluronate, the cleavage products included both the preponderant analogous repeating disaccharide multiples with N-acetyl-D-galactosamine at the reducing end and smaller quantities of oligosaccharides composed of an odd number of monosaccharides. These latter products were presumably derived from the ends of the shortened chondroitin chains, resulting from methanolysis during the desulfation and alkaline elimination of reducing hexosamines. CD spectroscopic analysis of chondroitin relative to its constituent monosaccharides showed that this glycosaminoglycan does not share the structural feature which results in substantially enhanced CD properties for hyaluronate. A hypothesis relating the CD properties of Nacetylated hexosamines in oligo- and polysaccharides to the dihedral angle about the carbon-oxygen bond at C3 may explain the CD dependence on both the hexosaminidic linkage and hexosamine configuration at C4.