Selective depolymerisation of heparin to produce radio-labelled substrates for sulfamidase, 2-acetamido-2-deoxy-alpha-D-glucosidase, acetyl-CoA:2-amino-2-deoxy-alpha-D-glucoside N-acetyltransferase, and 2-acetamido-2-deoxy-D-glucose 6-sulfate sulfatase.
Heparin was carboxyl-reduced with NaBT4, and degraded under conditions of acid hydrolysis that selectively cleaved the 2-0-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 leads to 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 0-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-(1 leads to 4)-L-[6-3H]idopyranose, 0-(2-amino-2-deoxy-alpha-D-glycopyranosyl)-(1 leads to 4)-L-[6-3H]idopyranose 2-sulfate, and 0-(2-amino-2-deoxy-alpha-D-glucopyranosyl 6-sulfate]-(1 leads to 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 0-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-(1 leads to 4)-L-[6-3H]idopyranose, the isolation of which suggests the presence of an IdA(OSO-3)-GlcNAc-IdA(OSO-3) 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 structures for some of these tetrasaccharides are proposed. Disaccharide and tetrasaccharide species were evaluated before and after N-acetylation or N-sulfation, as substrates for sulfamidase, acetyl-CoA: 2-amino-2-deoxy-alpha-D-glucoside N-acetyl-transferase, 2-acetamido-2-deoxy-alpha-D-glucosidase, or 2-acetamido-2-deoxy-D-glucose 6-sulfate sulfatase in human-skin fibroblasts.
D000118:Acetylglucosaminidase / Q000378:metabolism*; D000123:Acetyltransferases / Q000378:metabolism*; D004187:Disaccharides; D005944:Glucosamine / Q000378:metabolism; D006493:Heparin; D006596:Hexosaminidases / Q000378:metabolism*; D006867:Hydrolases / Q000378:metabolism*; D009844:Oligosaccharides; D011865:Radioisotope Dilution Technique; D013379:Substrate Specificity; D013429:Sulfatases / Q000378:metabolism*; D014316:Tritium
D004187:Disaccharides; D009844:Oligosaccharides; D014316:Tritium; D006493:Heparin; D000123:Acetyltransferases; C020580:glucosamine acetyltransferase; D006867:Hydrolases; D013429:Sulfatases; C018719:N-acetylglucosamine-6-sulfatase; C022708:N-sulfoglucosamine sulfohydrolase; D006596:Hexosaminidases; D000118:Acetylglucosaminidase; D005944:Glucosamine