Capillary electrophoresis determination of the binding affinity of bioactive sulfated polysaccharides to proteins: study of the binding properties of fucoidan to antithrombin.
The interaction of proteins with polysaccharides represents a major and challenging topic in glycobiology, since such complexes mediate fundamental biological mechanisms. An affinity capillary electrophoresis method has been developed to evidence the complex formation and to determine the binding properties between an anticoagulant polysaccharide of marine origin, fucoidan, and a potential target protein, antithrombin. This method is a variant of zonal electrophoresis in the mobility shift format. A fixed amount of protein was injected into a capillary filled with a background electrolyte containing the polysaccharide in varying concentrations. The effective mobility data of the protein were processed according to classical linearization treatments to obtain the binding constant for the polysaccharide/antithrombin complex. The results indicate that fucoidan binds to antithrombin in a 1:1 stoichiometry and with an affinity depending on the molecular weight of the polysaccharide. For heparin, the binding constant obtained similarly is in accordance with the literature. This is the first report showing the implementation of a capillary electrophoresis method contributing to the mechanistic understanding of the biological activities of fucoidan and providing evidence for the complex formation between fucoidan and the protein inhibitor of the coagulation antithrombin.
Varenne
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A
Laboratoire d'Electrochimie et Chimie Analytique, UMR CNRS 7575, ENSCP, 11, Rue Pierre et Marie Curie, 75231 Paris Cedex 05, France. varenne@ext.jussieu.fr
Gareil
P
P
Colliec-Jouault
S
S
Daniel
R
R
D000991:Antithrombins / Q000737:chemistry / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D056890:Eukaryota; D006493:Heparin / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D013431:Sulfates / Q000737:chemistry*; D013816:Thermodynamics
D000991:Antithrombins; D011134:Polysaccharides; D013431:Sulfates; D006493:Heparin; C007789:fucoidan