Selective depolymerisation of heparin to produce radio-labelled substrates for sulfamidase, 2-acetamido-2-deoxy-α-d-glucosidase, acetyl-CoA:2-amino-2-deoxy-α-d-glucoside N-acetyltransferase, and 2-acetamido-2-deoxy-d-glucose 6-sulfate sulfatase

Abstract

Heparin was carboxyl-reduced with NaBT4, and degraded under conditions of acid hydrolysis that selectively cleaved the 2-O-sulfo-l-idopyranosidic linkages. The resulting, radiolabelled-disaccharides and -tetrasaccharides were isolated by gel chromatography, and then fractionated by ion-exchange chromatography, paper chromatography, and paper electrophoresis. Of the nine disaccharides isolated and identified, eight were probably derived from the major repeating-disaccharide unit in heparin (2-deoxy-2-sulfoamino-d-glucosyl 6-sulfate → l-idosyluronic acid 2-sulfate). Sodium borotritide reduction and/or HNO2 deamination of these eight disaccharide fractions indicated four to contain l-idopyranose residues and the other four to contain 1,6-anhydro-l-idopyranose residues as terminal units. The latter, terminal unit probably represents a minor component formed during the acid hydrolysis. On the basis of N-acetylation, N-sulfation, and HNO2-deamination studies, and the known positions and configurations of the glycosidic and sulfate linkages in heparin, four disaccharides were identified as O-(2-amino-2-deoxy-α-d-glucopyranosyl)-(1→4)-l-[6-3H]idopyranose, O-(2-amino-2-deoxy-α-d-glucopyranosyl 6-sulfate)-(1→4)-l-[6-3H]idopyranose, O-(2-amino-2-deoxy-α-d-glucopyranosyl)-(1→4)-l-[6-3H]idopyranose 2-sulfate, and O-(2-amino-2-deoxy-α-d-glucopyranosyl 6-sulfate]-(1→4)-l-[6-3H]idopyranose 2-sulfate. A similar set of four disaccharides contained 1,6-anhydro-l-[6-3H]idopyranose residues in place of the l-[6-3H]idopyranose residues. The other disaccharide was tentatively identified as O-(2-acetamido-2-deoxy-α-d-glucopyranosyl)-(1→4)-l-[6-3H]idopyranose, the isolation of which suggests the presence or an {A figure is presented} sequence in the heparin preparation, which accounts for at least 1% of its total sequence. The tetrasaccharides were fractionated, on the basis of their sulfate content, into at least five species by ion-exchange chromatography or by paper electrophoresis. These were fractionated further into species with and without carboxyl groups, and with l-idopyranose or 1,6-anhydro-l-idopyranose residues as terminal units. Tentative. © 1980.