Binding of angiogenesis inhibitor kringle 5 to its specific ligands by frontal affinity chromatography

• The ligands bind to Kringle 5 on the lysine binding site in equimolar amounts.
• Hydrogen bond and Van der Waals force are the driving forces for the interaction.
• The binding affinity depends on the flexible feature of the ligands.

The interactions between angiogenesis inhibitor Kringle 5 and its five specific ligands were investigated by frontal affinity chromatography in combination with fluorescence spectra and site-directed molecular docking. The binding constants of trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCHA), epsilon-aminocaproic acid (EACA), benzylamine, 7-aminoheptanoic acid (7-AHA) and l-lysine to Kringle 5 were 19.0 × 103, 7.97 × 103, 6.45 × 103, 6.07 × 103 and 4.04 × 103 L/mol, respectively. The five ligands bound to Kringle 5 on the lysine binding site in equimolar amounts, which was pushed mainly by hydrogen bond and Van der Waals force. This binding affinity was believed to be dependent on the functional group and flexible feature in ligands. This study will provide an important insight into the binding mechanism of angiogenesis inhibitor Kringle 5 to its specific ligands.