Abstract
Heparin was cleaved with nitrous acid at pH 1.5 and the products were reduced with Na+ boro[3H]hydride to generate a mixture of di- and tetrasaccharides having anhydro-D-[3H]mannitol (AManR) residues on their reducing terminals. The products were purified to homogeneity by gel filtration and high-performance liquid chromatography. For each oligosaccharide, the proportions of D-glucuronic acid (GlcUA), L-iduronic acid (IdoUA), N-acetyl-D-glucosamine (GlcNAc), and AManR and the monosaccharide sequence were determined by quantification of the products of acid hydrolysis. The tetrasaccharide sequences were determined by comparison of the disaccharide units formed by hydrazinolysis and deamination with previously characterized disaccharides. The following new oligosaccharides were identified: GlcUA(2-SO4)-AManR, GlcUA(2-SO4)-AManR(6-SO4), GlcUA-AManR(3,6-diSO4), GlcUA-GlcNAc-GlcUA-AManR, IdoUA-GlcNAc-GlcUA-AManR, GlcUA-GlcNAc(6-SO4)-GlcUA-AManR, IdoUA(2-SO4)-GlcNAc-GlcUA-AManR, IdoUA-GlcNAc(6-SO4)-GlcUA-AManR, IdoUA(2-SO4)-GlcNAc-GlcUA-AManR(6-SO4), IdoUA-GlcNAc(6-SO4)-GlcUA-AManR(6-SO4), IdoUA-GlcNAc(6-SO4)-GlcUA-AManR(3-SO4), IdoUA-GlcNAc(6-SO4)-GlcUA-AManR(3,6-diSO4), and IdoUA(2-SO4)-GlcNAc(6-SO4)-GlcUA-AManR(6-SO4). Then the disaccharides and the tetrasaccharides were readily resolved by high-performance anion-exchange liquid chromatography and were quantified on the basis of the amount of 3H counts/min in each. The structures are discussed in terms of their implications regarding heparin biosynthesis and anticoagulant activity.
Highlights
Heparin was cleaved with nitrous acidat pH 1.5 and biosynthesis of heparin (2,3) suggest that its structure is the products were reduced with Na+ boro[’H]hydride further complicated by the fact that thedisaccharide sequence to generate a mixture odfi- and tetrasaccharideshav- may differ for each polymer chain in a heparin preparation
SO4). the disaccharides and the tetrasaccharides were readily resolved by high-performanceanion-ex- Chromatography and Electrophoresis-Radiolabeled compounds change liquid chromatography anwd ere quantified on were separated by descending paper chromatography in one of the the basis of the amount of ‘H counts/min in each
The following solvent systems: System 1: 1-butanol, glacial acetic acid, 1 structures are discussed in termsof their implications N ammonium hydroxide (2:3:1); System 2: 1-butanol, glacial acetic regarding heparin biosynthesisand anticoagulant ac- acid, 1 N ammonium hyroxide (2:3:1.5); System 3: 1-butanol, glacial tivity
Summary
Purification of Oligosaccharides-A mixture of NaB3H4- which had been characterized previously (14) It reduced oligosaccharides obtained from nitrous acid-treated contained three components which had not beenobserved hog mucosaheparin was separated accordingto size on a Bio- before. These were designated d l through d3. The composition and the analysis of the monosaccharide sequence were carried out by determining the amounts of the previously characterized mono- and disaccharides formed when the oligosaccharide was subjected to acid hydrolysis (29).As an example of such an analysis, Fig. 4 shows the progress of release of intermediates during the hydrolysis of tetrasaccharide t7. Analysis of the samples after rereduction with NaB3H4(data not presented) showed the appearance of the IdoUA which, due to the lability of the iduronosyl bond (29), was released rapidly from the nonreducing terminal, and additional
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