FIGURE 1.

Characterization of HS/heparin from WT and HS6ST-2^−/− ears. GAGs were prepared from the ears of 8-week-old WT and HS6ST-2^−/− mice as described under “Experimental Procedures.” A, aliquot of these GAGs was digested with a mixture of heparitinase-I, heparitinase-II, and heparinase and then subjected to reverse phase ion pair chromatography with postcolumn fluorescence labeling as described under “Experimental Procedures.” The histogram shows the percent composition of unsaturated disaccharides in HS/heparin from wild type (open bars) and HS6ST-2^−/− ears (filled bars). ΔDi-0S, ΔDi-NS, ΔDi-6S, ΔDi-(N,6)diS, ΔDi-(N,2)diS, and ΔDi-(N,6,2)triS are unsaturated disaccharides derived from the following units in HS/heparin-HexA-GlcNAc-, -HexA-GlcNSO₃-, -HexA-GlcNAc(6SO₄)-, -HexA-GlcNSO₃(6SO₄)-, -HexA(2SO₄)-GlcNSO₃-, and -HexA(2SO₄)-GlcNSO₃(6SO₄)-, respectively. The values shown are the means of triplicate analyses, and the bars represent the standard deviation. B, GAGs from wild type and HS6ST-2^−/− ears were digested with chondroitinase ABC; the digested products were removed, and the remaining HS/heparin from wild type and HS6ST-2^−/−ears were fractionated into the following six fractions by Mono Q column chromatography as described under “Experimental Procedures”: fraction I (30.5–33.5 min), fraction II (34–36.5 min), fraction III (37–41 min), fraction IV (41.5–44 min), fraction V (44.5–46 min), and fraction VI (46.5–49 min). The % yields of each fraction from WT and HS6ST-2^−/− heparin/HS were calculated from the total amounts of unsaturated disaccharides. C, disaccharide composition of each fraction was analyzed as described under “Experimental Procedures.”