SPECIFICITY STUDIES ON THE HEPARIN LYASES FROM FLAVOBACTERIUM-HEPARINUM

An understanding of the substrate specificity study of the heparin lya ses (heparinase and heparitinases) is crucial for elucidation of the s equence of heparin and heparan sulfate. Four chemically modified hepar ins have been used to study the substrate specificity of the three hep arin lyases. These modified heparins include the N- and O-desulfated a nd then specifically N-sulfated or N-acetylated derivatives of heparin and a modified heparin containing L-galactopyranosyluronic acid resid ues. These chemically modified heparins were degraded to various exten ts by the three heparin lyases. Differences in degree of sulfation hav e profound impact on the ease of cleavage of glycosidic linkages. Hepa rin lyase I (EC 4.2.2.7) is selective in cleaving highly sulfated poly saccharide chains containing linkages to 2-O-sulfated alpha-L-idopyran osyluronic acid residues. Heparin lyase III (EC 4.2.2.8) cleaves linka ges that have reduced density of sulfation and that contain beta-D-glu copyranosyluronic acid residues. The ability of heparin lyase III to a ct on linkages to unsulfated alpha-L-idopyranosyluronic acid residues is observed for the first time. Heparin lyase II (no assigned EC numbe r) demonstrates an unparalleled, wide specificity for substrates compr ised of linkages containing both alpha-L-idopyranosyluronic and beta-D -glucopyranosyluronic acid residues. Heparin lyase II can also act on substrates containing linkages to unnatural alpha-L-galactopyranosylur onic acid residues. The high level of specificity of heparin lyase I m akes it particularly suitable for use in the sequencing of heparin and heparan sulfate, while caution must be excercised in using heparin ly ases II and III to sequence heparin and heparan sulfate because of the ir relatively broad specificity.