Heparin was converted by treatment with nitrous acid primarily into sulfated disaccharides. The mixture of disaccharides was reduced with sodium boi@H]hydride and the disaccharides were purified by preparative paper electrophoresis and paper chromatography. Four disaccharides were obtained. On the basis of their paper electrophoretic mobilities and the products formed at intermediate stages of their acid hydrolysis, the disaccharides were identified as 4-O-(2-O-sulfa-a-L-idopyranosyluronic acid)&O-sulfo-2,5-anhydro-o-mannitol, 4-0-(2-0-sulfo-a-L-idopyranosyluronic acid)-2,5-anhydro-o-mannitol, 4-0-(a+idopyranosyluronic acid)-6-0-sulfo2,5-anhydro-o-mannitol, and 4-O-(8 D-glucopyranosyluronic acid)&-0-sulfo-2,5-anhydro-o-mannitol.
The purified disaccharides were used as standards in the development of a high-performance liquid chromatography procedure for their separation and quantitation on a Partisil-10 SAX anion-exchange column. The three monosulfated disaccharides were resolved by isocratic elution with 40 mM KH,PO,. The KHZPOI concentration was then increased to 400 mM to elute the disulfated disaccharide. Column effluents were collected in M-ml fractions, and the recovery of each 3H-labeled product was determined by scintillation counting. When sodium boro-[3H]hydride with a specific activity of 315 mCi/mmol was used in the reduction of the heparin deamination products, the disaccharides gave 28,500 cpm/nmol in the effluent peaks. Quantitative recoveries of the 3H-disaccharides were obtained. It was demonstrated that the method developed using the purified disaccharides gave reproducible and quantitative results in direct assays of aliquots of boro[3H]hydride-reduced heparin deamination mixtures.
Heparin is composed of alternating Dglucosamine (GIcN)~ and uranic acid residues. Most of the D-glucosamine residues are N-sulfated, but some are N-acetylated. Either D-glucuronic acid (GlcU) or Liduronic acid (IdU) may appear in the uranic acid positions in the polymer, with IdU constituting from 70 to 90% of the total uranic