Heparinase II from Flavobacterium heparinum

Saccharides produced by the action of heparinase II on native pig mucosal heparin (heparin I s ), de‐ N ‐sulphated heparin (heparin I h ), N ‐acetylheparin (heparin I a ), de‐ N/O ‐sulphated heparin (heparin IV h ), de‐ O ‐sulphated heparin (heparin IV s ) and de‐ O ‐sulphated N ‐acetylheparin (heparin IV a ) were analysed by reversed‐phase HPLC using Spherisorb ODS2. Fractions obtained by gel filtration with Bio‐Gel P‐4 were similarly examined. Heparin I s gave ΔUA‐2S → GlcNS‐6S (I s ) as the major unsaturated disaccharide and lesser amounts of ΔUA → GlcNS‐6S (II s ), ΔUA‐2S → GlcNS (III s ), ΔUA → GlcNS (IV s ), ΔUA‐2S → GlcNAc‐6S (I a ), ΔUA → GlcNAc‐6S (II a ), ΔUA‐2S → GlcNAc (III a ) and ΔUA → GlcNAc (IV a ). Heparins I a , IV a and IV s gave as the predominant unsaturated disaccharide that corresponding to the major repeat structure of the polymer. These were respectively ΔUA‐2S → GlcNAc‐6S (I a ), ΔUA → GlcNAc (IV a ) and ΔUA → GlcNS (IV s ). Minor disaccharides from the heterogeneous structure in native pig heparin and from residual O ‐sulphates after the de‐ O ‐sulphating process were detected. Heparin I h was degraded more slowly than any of the N ‐substituted heparins. The predominant unsaturated disaccharide was I h , which was derived from the major repeating unit. In addition, disaccharides II h , III h , I a , II a and IV a were detected. Heparin IV h showed little degradation, the unsaturated disaccharide IV h not being detected after 24 h. Disaccharide IV a was obtained from the heterogeneous sequence in heparin IV h . Several higher oligosaccharides were identified in the gel‐filtration fractions including saccharides from the linkage region (for heparin I s and IV a ) and the anti‐thrombin binding site (for heparin I s only). A tetrasaccharide and hexasaccharide, with the structures ΔUA → GlcNAc → UA → GlcNAc and ΔUA → GlcNAc → UA → GlcNAc → UA → GlcNAc, were present in the HPLC profiles of heparins I a and IV a .