Structural elucidation of glycosaminoglycans through characterization of disaccharides obtained after fragmentation by hydrazine-nitrous acid treatment.
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.
D000818:Animals; D001894:Borohydrides; D055598:Chemical Phenomena; D002621:Chemistry; D002809:Chondroitin Sulfates / Q000032:analysis; D002855:Chromatography, Thin Layer; D003315:Cornea / Q000032:analysis; D003871:Dermatan Sulfate / Q000032:analysis; D004187:Disaccharides / Q000032:analysis*; D006025:Glycosaminoglycans / Q000032:analysis*; D006493:Heparin / Q000032:analysis; D006801:Humans; D006820:Hyaluronic Acid / Q000032:analysis; D006834:Hydrazines; D007231:Infant, Newborn; D007632:Keratan Sulfate / Q000032:analysis; D008353:Mannitol / Q000031:analogs & derivatives / Q000302:isolation & purification; D009608:Nitrous Acid; D012754:Sharks; D013402:Sugar Alcohols / Q000302:isolation & purification; D013431:Sulfates / Q000378:metabolism; D013552:Swine; D014907:Whales
D001894:Borohydrides; D004187:Disaccharides; D006025:Glycosaminoglycans; D006834:Hydrazines; D013402:Sugar Alcohols; D013431:Sulfates; D003871:Dermatan Sulfate; C029424:hydrazine; D008353:Mannitol; C031765:2,5-anhydromannitol; C046108:2,5-anhydrotalitol; C025364:sodium borohydride; D006820:Hyaluronic Acid; D006493:Heparin; D002809:Chondroitin Sulfates; D007632:Keratan Sulfate; D009608:Nitrous Acid