Enzymatic Preparation of Heparin Oligosaccharides Containing Antithrombin III Binding Sites

Authors
Toida, Toshihiko
Hileman, Ronald E.
Smith, April E.
Vlahova, Petinka I.
Linhardt, Robert J.
ORCID
https://orcid.org/0000-0003-2219-5833
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Issue Date
1996-12-30
Keywords
Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
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Full Citation
Enzymatic Preparation of Heparin Oligosaccharides Containing Antithrombin III Binding Sites, T. E. Toida, R. E. Hileman, A. E. Smith, P. I. Vlahova, R.J. Linhardt, Journal of Biological Chemistry, 271, 32040-32047, 1996.
Abstract
Two new oligosaccharides were prepared from heparin by its partial depolymerization using heparin lyase I (EC 4.2.2.7) in an attempt to prepare oligosaccharides having intact antithrombin III binding sites. The oligosaccharides were purified by chromatography on the basis of both size and charge and demonstrated a high level of purity by capillary electrophoresis. One- and two-dimensional 1H NMR spectroscopy at 500 MHz revealed the structure of each oligosaccharide. The octasaccharide and decasaccharide are ΔUAp2S(1→4)-α-DGlcNpS6S(1→4)-α-L-IdoAp(1→4)-α-D-GlcNpAc6S(1→4)-βD-GlcAp(1→4)-α-D-GlcNpS3S6S(1→4)-α-L-IdoAp2S(1→4)α-D-GlcNpS6S (where ΔUAp is 4-deoxy-α-L-threo-hex-enopyranosyluronic acid, GlcNp is 2-amino-2-deoxy-glucopyranose, GlcAp is glucopyranosyluronic acid, S is sulfate and Ac is acetate) and ΔUAp2S(1→4)-α-D-GlcNpS6S(1→4)-α-L-IdoAp(1→4)-α-D-GlcNpAc6S (1→4)-β-D-GlcAp(1→4)-α-D-GlcNpS3S6S(1→4)-α-L-IdoAp2S(1→4)-α-D-GlcNpS6S(1→4)-α-L-IdoAp2S(1→4)-α-D-GlcNpS6S, respectively. A hexasaccharide containing a similar structural motif to that found in the antithrombin III binding site and having greatly reduced anticoagulant activity was also isolated. The structure of the hexasaccharide is ΔUAp2S(1→4)-α-D-GlcNpAc6S(1→4)-β-D-GlcAp(1→4)-α-D-GlcNpS3S6S(1→4)-α-L-IdoAp(1→4)-α-D-GlcNpS6S. The octasaccharide and decasaccharide correspond to the predominant structural motif found in porcine intestinal mucosal heparin. Sufficient quantities of the decasaccharide were obtained to examine its interaction with antithrombin III using microtitration calorimetry. This decasaccharide bound to antithrombin III with similar avidity as heparin and showed comparable anticoagulant activity, as determined using an antithrombin III dependent anti-factor Xa assay. Interestingly, while both decasaccharide and heparin bound to antithrombin with nanomolar affinity, very little heat of binding was observed.
Description
Journal of Biological Chemistry, 271, 32040-32047
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Department
The Linhardt Research Labs.
The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
Publisher
Elsevier
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The Linhardt Research Labs Online Collection
Rensselaer Polytechnic Institute, Troy, NY
Journal of Biological Chemistry
https://harc.rpi.edu/
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