Binding of fibrin monomer and heparin to thrombin in a ternary complex alters the environment of the thrombin catalytic site, reduces affinity for hirudin, and inhibits cleavage of fibrinogen.

Interaction of the blood clotting proteinase, thrombin, with fibrin monomer and heparin to form a thrombin.fibrin monomer.heparin ternary complex is accompanied by a change in thrombin catalytic specificity. Equilibrium binding interactions in the assembly of the ternary complex were characterized quantitatively using thrombin labeled at the active site with a fluorescent probe and related to changes in thrombin specificity toward exosite I-dependent binding of hirudin and cleavage of fibrinogen. Changes in the active site environment accompanying binding of heparin or fibrin to thrombin in binary complexes were reported by fluorescence enhancements which contributed additively to the perturbation accompanying formation of the ternary complex. Quantitative analysis of the interactions supports a preferentially ordered path of ternary complex assembly, in which initial binding of heparin to thrombin facilitates binding of fibrin monomer with an approximately 40-fold increased affinity. Binding of fibrin monomer in the ternary complex decreased the affinity of native thrombin for hirudin by >100-fold and inhibited cleavage of fibrinogen, but this inhibition was overcome when fibrin(ogen)-fibrin interactions occurred. These results support a ternary complex model in which heparin binding through exosite II of thrombin facilitates fibrin monomer binding via exosite I, with accompanying changes in thrombin catalytic specificity resulting from perturbations in the active site and reduced accessibility of exosite I to hirudin and fibrinogen.

Modeling of the electrostatic potential field of plastocyanin.

The DelPhi computer program is used to calculate the electrostatic potential field of the photosynthetic electron transport protein plastocyanin. Knowledge of the potential field is important for understanding the mechanisms by which plastocyanin interacts with other charged reagents. The program uses a macroscopic, continuum approach in which the protein and solvent are assigned different dielectric constants, the crystal structure of the protein defines the dielectric boundary, and the ionic strength of the solvent is taken into account. The potential field is determined by numerically solving the Poisson-Boltzmann equation. The field surrounding plastocyanin is characterized by a region of positive potential over the copper center active site, and a region of negative potential over the adjacent association site containing tyrosine 83. The shape and magnitude of the potential field shows a strong dependence on the ionic strength and pH of the solvent. The program is able to accurately predict the effect of the copper center oxidation state on the pKa of a tetranitromethane derivative of tyrosine 83 using an intrinsic protein dielectric constant of 2 to 4. Evidence is also presented that the glutamate 68 side chain is exposed to the solvent to a greater extent in the solution structure of plastocyanin than in the crystal structure.