Contribution of 3-O- and 6-O-sulfated glucosamine residues in the heparin-induced conformational change in antithrombin III
- 1 October 1987
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 26 (20), 6454-6461
- https://doi.org/10.1021/bi00394a024
Abstract
The role of 3-O- and 6-O sulfated glucosamine residues within the heparin octasaccharide critical for biological activity, iduronic acid(1) .fwdarw. N-acetylglucosamine 6-O sulfate(2) .fwdarw. glucuronic acid(3) .fwdarw. N-sulfated glucosamine 3,6-di-O-sulfate(4) .fwdarw. iduronic acid 2-O-sulfate(5) .fwdarw. N-sulfated glucosamine 6-O-sulfate(6) .fwdarw. iduronic acid 2-O-sulfate(7) .fwdarw. anhydromannitol 6-O-sulfate(8), was determined by comparing its ability to bind antithrombin, induce a conformational change in this protease inhibitor as monitored by the enhancement of intrinsic flourescence, and accelerate (at saturation) the interaction of this protein with human factor Xa. The octasaccharide produced a maximum 48% increase in intrinsic fluorescence 37.degree. C and a rate of factor Xa inhibition of 6 .times. 105 M-1 s-1 as measured by stopped-flow fluorometry at 25.degree. C. The basal rate of the antithrombin-factor Xa interaction observed in the absence of oligosaccharide was 2 .times. 103 M-1 s-1. The synthetic pentasaccharide, consisting of residues 2-6, produced fluorescence enhancement and rate of inhibition equal to those of the octasaccharide. However, a similar pentasaccharide, identical in all respects except that it lacked the 3-O-sulfate on residue 4, produced less than a 5% fluoresence enhancement and a rate of factor Xa inhibition of 8 .times. 103 M-1 s-1. The tetrasaccharide consisting of residues 2-5 produced a 35% fluorescence enhancement and a rate of factor Xa inhibition 3 .times. 105 M-1 s-1. The terasaccharide consisting of residues 3-6 produced a 33% fluorescence enhancement and a rate of factor Xa inhibition of 6 .times. 104 M-1 s-1. Thus, the loss of either the 6-O-sulfated residue 2 or the 3-O-sulfate of residue 4 results in a 30-95% loss in the ability of the pentasaccharide to enhance the intrinsic fluorescence of antithrombin and a 10-76-fold reduction in the rate of factor Xa inhibition. These two residues must, necessarily, represent the major contributors to a conformational change in antithrombin, which is linked to the biological activity of the octasaccharide. In addition, nonproportional changes in the intrinsic flouresence enhancement and the acceleration of factor Xa neutralization indicate that multiple conformational stages can occur in antithrombin when complexed to these oligosaccharides.This publication has 28 references indexed in Scilit:
- The binding of low molecular weight heparin to hemostatic enzymes.Journal of Biological Chemistry, 1980
- The kinetics of hemostatic enzyme-antithrombin interactions in the presence of low molecular weight heparin.Journal of Biological Chemistry, 1980
- Evidence for a 3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of heparin.Proceedings of the National Academy of Sciences, 1980
- A novel 3-0 sulfatase from human urine acting on methyl-2-deoxy-2-sulfamino-α-D-glucopyranoside 3-sulfateBiochemical and Biophysical Research Communications, 1980
- Structure of the antithrombin-binding site in heparin.Proceedings of the National Academy of Sciences, 1979
- Correlation between structure and function of heparinProceedings of the National Academy of Sciences, 1979
- Structure-function relationships of heparin speciesProceedings of the National Academy of Sciences, 1978
- The Size and Shape of Human and Bovine Antithrombin IIIEuropean Journal of Biochemistry, 1977
- The separation of active and inactive forms of heparinBiochemical and Biophysical Research Communications, 1976
- A modified uronic acid carbazole reactionAnalytical Biochemistry, 1962