SKELETAL KERATAN SULFATE STRUCTURAL-ANALYSIS USING KERATANASE-II DIGESTION FOLLOWED BY HIGH-PERFORMANCE ANION-EXCHANGE CHROMATOGRAPHY

A semi-quantitative fingerprinting method has been developed for the structural analysis of skeletal keratan sulphates. This involves the digestion of the parent keratan sulphate chains with the enzyme keratanase II (Bacillus sp,), followed by reduction of the resulting oligosaccharides with sodium borohydride and chromatography on a Dionex AS4A-SC column. This column has been calibrated for the elution positions of 26 previously characterized oligosaccharides (Brown et al., Biochemistry, 33, 4836-4846, 1994; Brown et al., Ear. J. Biochem., 224, 281-308, 1994). The technique permits sample analysis with pulsed electrochemical detection (sensitive to similar to 5 ng of oligosaccharide) or by monitoring [H-3] or [S-35] radiolabel (potentially sensitive to similar to 1 pg or less of an oligosaccharide); thus permitting the study of sub-microgram amounts of keratan sulphates. Skeletal keratan sulphates from a number of sources have been examined in this chromatographic system and their structural features are discussed.