The disaccharide composition of heparins and heparan sulfates

Abstract

Heparin and heparan sulfate can be cleaved selectively at their N-sulfated glucosamine residues by direct treatment with nitrous acid at pH 1.5. These polymers can also be cleaved selectively at their N-acetylated glucosamine residues by first N-deacetylating with hydrazine and then treating the products with nitrous acid at pH 4. These procedures have been combined and optimized for the conversion of these glycosaminoglycan chains into their disaccharide units. A modified hydrazinolysis procedure in which the glycosaminoglycans were heated with hydrazine:water (70:30) containing 1% hydrazine sulfate gave rapid rates of N-deacetylation and minimal conversion of the uronic acid residues to their hydrazide derivatives. Under these conditions, N-deacetylation was complete in 4 h and the β-eliminative cleavage of the polymer chains that occurs during hydrazinolysis ( P. N. Shaklee and H. E. Conrad (1984) Biochem. J. 217, 187–197 ) was eliminated. Treatment of the N-deacetylated polymer with nitrous acid at pH 3 for 15 h at 25°C then gave simulataneous cleavage at the N-unsubstituted glucosamine residues and the N-sulfated glucosamine residues. These deamination conditions minimized, but did not eliminate, the side reaction in which nitrous acidreactive glucosamine residues undergo ring contraction without glucosaminide bond cleavage. Thus, the disaccharides were obtained in a yield of 90% of those originally present in the glycosaminoglycan chains. Since the ring contraction side reaction occurs randomly at the diazotized glucosamine residues, the disaccharides formed in the pH 3 nitrous acid reaction were recovered in proportions equal to those in the original glycosaminoglycan chain. Anion-exchange high-pressure liquid chromatography analysis of the disaccharides ( M. J. Bienkowski and H. E. Conrad (1985) J. Biol. Chem. 260, 356–365 ) then gave quantitative analyses of the amounts of each disaccharide type in the heparinoid chains.