Gm. Brown et al., SKELETAL KERATAN SULFATE STRUCTURAL-ANALYSIS USING KERATANASE-II DIGESTION FOLLOWED BY HIGH-PERFORMANCE ANION-EXCHANGE CHROMATOGRAPHY, Glycobiology, 5(3), 1995, pp. 311-317
A semi-quantitative fingerprinting method has been developed for the s
tructural analysis of skeletal keratan sulphates. This involves the di
gestion of the parent keratan sulphate chains with the enzyme keratana
se II (Bacillus sp,), followed by reduction of the resulting oligosacc
harides 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., Biochemist
ry, 33, 4836-4846, 1994; Brown et al., Ear. J. Biochem., 224, 281-308,
1994). The technique permits sample analysis with pulsed electrochemi
cal detection (sensitive to similar to 5 ng of oligosaccharide) or by
monitoring [H-3] or [S-35] radiolabel (potentially sensitive to simila
r to 1 pg or less of an oligosaccharide); thus permitting the study of
sub-microgram amounts of keratan sulphates. Skeletal keratan sulphate
s from a number of sources have been examined in this chromatographic
system and their structural features are discussed.