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Title:Analysis of the Structural Units of Sulfated Glycosaminoglycans by High Performance Ion Exchange Chromatography
Author(s):Delaney, Stephen Ralph
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Chemistry, Biochemistry
Abstract:The structural units of heparin produced by nitrous acid deamination were stoichiometrically reduced with NaB('3)H(,4) and analyzed by high performance ion exchange chromatography to obtain resolution of the four primary disaccharide units of heparin; 4-O-((beta)-D-glucopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-D-mannitol (GMS), two-L-iduronic acid-containing monosulfated disaccharides, 4-O-((alpha)-L-idopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-D-mannitol (IMS) and 4-O-(2-O-sulfo-(alpha)-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol (ISM), and a single disulfated disaccharide, 4-O-(2-O-sulfo-(alpha)-L-idopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-D-mannitol (ISMS). The capacity to separate tetrasaccharides or higher oligosaccharides was demonstrated using chondroitin sulfate oligosaccharides which could be structurally characterized in terms of their chondroitinase ABC digestion products. The high performance ion exchange chromatography technique has been used in the analysis of the oligosaccharide composition of a series of heparins fractionated by Dowex anion exchange chromatography and in the isolation and characterization of unidentified deamination oligosaccharides which appear in smaller amounts in the hplc profiles.
The analyses of the Dowex-fractionated heparins showed that the monosulfated disaccharide ISM and other uncharacterized monosulfated oligosaccharides are the major oligosaccharide units in Dowex fractions which elute in low salt. In fractions eluting at high ionic strength, the disulfated disaccharide, ISMS and the trisulfated tetrasaccharide involved in the specific binding of heparin to antithrombin III binding site are the main structural units in the deamination products of these fractions. The increase in the antithrombin III binding sequence determined by hplc analysis in these latter fractions correlates well with the increases in anticoagulant activities observed upon fractionation.
Isolation and characterization of unidentified oligosaccharide products in these deamination populations showed the presence of a monosulfated (with D-glucuronic acid reducing terminal) and a disulfated trisaccharide {(alpha)-L-idopyranosyluronic acid-(1(--->)4)-2-acetamido-2-deoxy-(alpha)-D-glucopyranose-(1(--->)4)-D-glucuronic acid}, a trisulfated tetrasaccharide {(alpha)-L-idopyranosyluronic acid-(1(--->)4)-2-acetamido-2-deoxy-(alpha)-D-glucopyranose-(1(--->)4)-(beta)-D-glucopyranosyluronic acid-(1(--->)4)-2,5-anhydro-D-mannitol}, and oligosaccharides whose monosaccharide sequences were only partially characterized. These findings suggest that a far greater structural heterogeneity exists in the linear sequence of heparin monosaccharide units than currently recognized.
Issue Date:1981
Type:Text
Language:English
Description:220 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1981.
URI:http://hdl.handle.net/2142/67417
Other Identifier(s):(UMI)AAI8203441
Date Available in IDEALS:2014-12-14
Date Deposited:1981


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