Electrostatic interactions in the heparin-enhanced reaction between human thrombin and antithrombin

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Abstract
Binding of heparin to thrombin is monitored by means of an aqueous two-phase partition system, and binding of heparin to antithrombin is monitored by means of heparin induced enhancement of the intrinsic fluorescence of the protein. Both types of binding are studied at various electrolyte compositions of the medium. Heparin is displaced from thrombin at lower concentrations of electrolyte than those necessary for its displacement from antithrombin. K+ is more efficient than Na+, which is again more efficient than Li+ in displacing heparin from these proteins. The kinetics of the reaction between thrombin and antithrombin in the presence of heparin were studied by using an assay where synthetic peptide substrate is present in the reaction mixture during the reaction between proteinase and inhibitor. The kinetics are studied at various electrolyte compositions of the medium and the results are compared with those obtained from the binding studies performed under similar conditions. The results are consistent with a model where binding of heparin to antithrombin causes enhancement of the reaction rate, and where this enhancement is abolished again when additional binding of heparin to thrombin takes place on further addition of heparin.