The sulfated galactan from de red alga B. occidentalis is a potent anticoagulant, with an activity of 130UI/mg when compared with heparin standard (180UI/mg). When tested in assays using specific proteases and coagulation inhibitors, the sulfated galactan and heparin show almost similar activity in assays using thrombin as the target protease and antithrombin as inhibitor. However, sulfated galactan and heparin differ in their preferred pathway of polysaccharide-activated inactivation of thrombin due to its binding with high affinity to protease but not to antithrombin. This proposal was based on changes in the intrinsic and extrinsic fluorescence of proteases promoted by sulfated polysaccharides binding. The titration of thrombin and antithrombin with the hidrophobic probe bis-ANS, leads to progressive increase in bis-ANS emission ad decrease in intrinsic fluorescence protein dual to the quenching effect exerted by bis-ANS on tryptophan emission. The addition of increasing concentrations of sulfated polysaccharides releases bis-ANS previously bind to the protein. Then, interaction of sulfated polisaccharides with protein can be follow by a decrease in bis-ANS emission and a recovery of intrinsic protein fluorescence. The changes in the bis-ANS and protein fluorescence were analyzed using computer fitting to estimate the dissociation constant for the binding of polysaccharides. The Kd for the binding of sulfated galactan and heparin to thrombin is 28nM and 61nM, respectively. These results suggest that the sulfated galactan and heparin interact with thrombin with hight affinity. In contrast, they differ markedly on their binding to antithrombin.

Financial support: CNPq, CAPES and FAPERJ.