1H NMR spectroscopic studies on the interactions between human plasma antithrombin III and defined low molecular weight heparin fragments.
The effects of length and composition upon the antithrombin-binding properties of heparin have been investigated for two series of structurally related heparin oligosaccharides. Each series consists of a tetrasaccharide, hexasaccharide, and octasaccharide heparin fragment composed of alternating hexuronic acid (either iduronate 2-sulfate or glucuronate) and glucosamine 6,N-disulfate residues. These two series represent dominant structural motifs in intact heparin and differ from each other by the presence of a glucuronic acid in one series in place of an iduronate 2-sulfate residue penultimate to the reducing end of the fragment. Perturbations to the 1H resonances in the NMR spectrum of antithrombin upon binding of the two series of heparin fragments are compared to those generated by intact heparin binding, as well as to the effects of binding of a synthetic high-affinity pentasaccharide. All of the heparin fragments examined appear to bind to antithrombin at the same site. Three of the heparin fragments (hexasaccharide-2, octasaccharide-2, and octasaccharide-1) produce almost identical perturbations in the antithrombin 1H NMR spectrum compared to binding of intact heparin, including perturbations of resonances from tryptophan 49. This indicates that neither the glucuronic acid nor the trisulfated glucosamine residue (structural elements known to be part of the high-affinity heparin motif) are necessary for the majority of the conformational changes induced upon heparin fragment binding to antithrombin. However, the low anticoagulant activity of these fragments indicates that the changes in protein conformation upon fragment binding, as manifested by these 1H resonance perturbations, are not sufficient for catalytic activation of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)
Horne
A
A
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Gettins
P
P
D000818:Animals; D000990:Antithrombin III / Q000737:chemistry*; D001665:Binding Sites; D002236:Carbohydrate Conformation; D006495:Heparin, Low-Molecular-Weight / Q000737:chemistry*; D006801:Humans; D007413:Intestinal Mucosa / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D009844:Oligosaccharides / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013552:Swine
D006495:Heparin, Low-Molecular-Weight; D009844:Oligosaccharides; D000990:Antithrombin III