Analysis of ligand-binding to the kringle 4 fragment from human plasminogen

The interaction of the isolated human plasminogen kringle 4 with the four omega-amino acid ligands epsilon-aminocaproic acid (epsilon ACA), N alpha-acetyl-L-lysine (AcLys), trans-aminomethyl(cyclohexane)carboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) has been further characterized by 1H-NMR spectroscopy at 300 and 600 MHz. Pronounced high-field shifts, reaching approximately 3 ppm, are observed for AMCHA resonances upon binding to kringle 4, which underscores the relevance of ligand lipophilic interactions with aromatic side chains at the binding site. Ligand titration curves for the nine His and Trp singlets found in the kringle 4 aromatic spectrum reveal a striking uniformity in the kringle response to the various ligands. The average binding curves exhibit a clear Langmuir absorption isotherm saturation profile and the data were analyzed under the assumption of one (high affinity) binding site per kringle. Equilibrium association constants (Ka) and first order dissociation rate constants (k off) were derived from linearized expressions of the Langmuir isotherm and of the spectral line-shapes, respectively. The results for the four ligands, at approximately 295 K, pH 7.2, indicate that: AMCHA exhibits the strongest binding (Ka = 159 mM-1) and epsilon ACA the weakest (Ka = 21 mM-1) with AcLys and BASA falling in between; epsilon ACA dissociates readily (k off = 5.3 X 10(3) s-1) and AMCHA associates the fastest (k on = 2.0 X 10(8) M-1 s-1) while the kinetics for BASA exchange is relatively slow (k off = 0.8 X 10(3) s-1, k on = 0.6 X 10(8) M-1 s-1); the ligand-binding kinetics is close to diffusion-controlled.