| Abstract: | Hydrazinolysis of glycosaminoglycans to bring about N-deacetylation followed by nitrous acid treatment to effect deaminative cleavage at alternating hexosamine residues has been used to make possible identification and quantitation of disaccharide sequences and position of O-sulfate substitution in nanogram amounts of these polymers. After radiolabeling by NaB3H4 reduction the hydrazine-nitrous acid products were fractionated on Dowex 1 and further resolved by thin-layer chromatography into disaccharides terminating in either sulfated or unsulfated anhydromannitol or anhydrotalitol. Fragmentation of hyaluronic acid, keratan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and heparin yielded a total of 14 disaccharides comprising the major sequences (greater than 1 mol%) occurring in mammalian glycosaminoglycans. Disaccharides representing the predominant variants of the chondroitin sulfates GlcUA beta 1----3anhydrotalitol(4-SO4) and GlcUA beta 1----3anhydrotalitol(6-SO4)] as well as of dermatan sulfate chains IdUA alpha 1----3anhydrotalitol(4-SO4) and GlcUA beta 1----3anhydrotalitol(4-SO4)] chains could readily be quantitated by this approach. In the case of heparin a comparison of the disaccharides produced by direct nitrous acid and hydrazine-nitrous acid treatments moreover provided an assessment of the distribution of N-sulfate groups. The characterization of the various disaccharides by Smith periodic acid degradation and glycosidase digestions was facilitated by the preparation and thin-layer chromatographic resolution of the complete series of monosulfated derivatives of anhydromannitol and anhydrotalitol; the sulfate esters were shown to be stable to both the hydrazine and nitrous acid treatments. The high sensitivity of the hydrazine-nitrous acid fragmentation procedure should prove useful in the structural elucidation of cell surface and basement membrane proteoglycans as well as other sulfated glycoconjugates which are present in small amounts. |
