The binding of tranexamic acid to native (Glu) and modified (Lys) human plasminogen and its effect on conformation.

The binding of 14C-labeled tranexamic acid (trunsAMCHA, transamin) to native (NH2 terminus Glu) and proteolytically modified (NH2 terminus Lys) human plasminogen was studied by an ultrafiltration technique, as was done earlier for the binding of e-aminocaproic acid to the same zymogen forms. Binding was determined at total ligand concentrations ranging from 5 pM to 16 mu, at pH 8, and 20°C. The binding constants were determined by a computer program using nonlinear least squares fitting techniques. In native plasminogen, tranexamic acid has a single strong binding site (&iss = 1.1 PM) and a group of about 4 weak sites (&ss = 0.75 I’fIM). In modified plasminogen, the first site is weaker than in the native one (Kdiss = 2.2 PM), but the second site is considerably stronger (Kdiss = 36 PM) than the corresponding one in the native protein. The remaining 2 to 3 sites in the modified zymogen bind tranexamic acid weakly (Kdiss = 1 mu). The conformational transition, known to accompany binding of this ligand to the native protein, was studied by sedimentation velocity measurements. A midpoint of 0.78 mu tranexamic acid was obtained for the effect, corresponding to the saturation of the weak set of sites, as was found earlier for l -aminocaproic acid binding. A much smaller change in sedimentation coefficient was also observed upon saturation of the modified zymogen. Correlation of binding and sedimentation data with rates of urokinase activation of the two proteins indicated that the strong binding of the first ligand molecule causes little change in the conformation of the protein and inhibits the activation rate only slightly. Saturation of the weak sites, however, is responsible for most of the conformational change and also for the large increase in activation rate of the native zymogen. Comparison of the binding constants, obtained in this study at homologous binding regions in the two proteins, permitted the calculation of free energy changes involved in the conformational transition in each of these binding regions.