Heparinase Digestion of 3- O -Sulfated Sequences: Selective Heparinase II Digestion for Separation and Identification of Binding Sequences Present in ATIII Affinity Fractions of Bovine Intestinal Heparins

Binding to antithrombin-III (ATIII) determines the anticoagulant activity of heparin. The complexes formed between heparin and ATIII result from a specific pentasaccharide sequence containing a 3- sulfated glucosamine in medium position. Building block analysis of heparins, following heparinase digestion, is a critical method in quality control that provides a simple structural characterization of a complex product. Hence, in these applications, study of the digestion of 3- -sulfated moieties merits special attention. With heparinase II, specific inhibition of cleavage of the non-reducing bond of 3- -sulfated units is observed. This specificity was erroneously generalized to other heparinases when it was observed that in exhaustive digests of heparins with the heparinase mixture, resistant 3- -sulfated tetrasaccharides were also obtained from the specific ATIII-binding pentasaccharides. In fact, the detection of unsaturated 3- -sulfated disaccharides in digests of heparin by heparinases I+II+III, resulting from the cleavage of the 3- sulfated unit by heparinase I in non-conventional sequences, shows that this inhibition has exceptions. Thus, in experiments where heparinase II is selectively applied, these sequences can only be digested into tetra- or hexasaccharides where the 3- -sulfated glucosamine is shifted on the reducing end. Heparinase I+II+III and heparinase II digests with additional tagging by reductive amination with sulfanilic acid were used to study the structural neighborhood of 3- -sulfated disaccharides in bovine mucosal heparin fractions with increasing affinity for ATIII. The 3- -sulfated disaccharides detected in heparinase I+II+III digests turn into numerous specific 3- -sulfated tetrasaccharides in heparinase II digests. Additionally, ATIII-binding pentasaccharides with an extra 3- -sulfate at the reducing glucosamine are detected in fractions of highest affinity as heparinase II-resistant hexasaccharides with two consecutive 3- -sulfated units.