Fluorescence and circular dichroism studies during the interactions of sulfated polysaccharides with antithrombin III

The changes in relative fluorescence of antithrombin III (AT-III) during its interaction with sulfated xylans were compared with that of sulfated glycosaminoglycans by measuring the ratio of the increase in fluorescence of AT-III in the presence of sulfated polysaccharide to the fluorescence of AT-III alone for various mass ratios. Interactions of corn cob xylan sulfate (CCXS) and sodium pentosan polysulfate (SP-54) with AT-III resulted in enhancements of relative fluorescence which were lower than commercial heparin. At mass ratios below 1, heparan sulfate and low molecular weight heparin (LMWH) gave increases in the relative fluorescence higher than that of commercial heparin, while highly sulfated semisynthetic chondroitin sulfates A and C gave much smaller increases. The relative fluorescence enhancements of AT-III by heparan sulfate, commercial heparin, LMWH and heparin derived pentasaccharide (HDP) increased with increasing mass ratios while the enhancements by CCXS, SP-54 and the highly sulfated chondroitin sulfates A and C were reversed at higher mass ratios. The estimated dissociation constants (kd) for the interaction of AT-III and the heparin-related compounds showed that heparin sulfate and LMWH gave the lowest kd values indicating a higher affinity for AT-III while commercial heparin and HDP gave higher kd values, indicating a lower affinity for AT-III. SP-54 gave a kd value lower than CCXS, indicating a greater affinity for AT-III. A comparison of the near ultraviolet (UV) circular dichroism (CD) spectrum of AT-III alone and during its interaction with oat spelts xylan sulfate (OSXS) showed enhancements of the two aromatic amino acid regions corresponding to phenylalanine and tryptophan.