Binding constant of VIVO to transferrin

The interactions of bis-(VO)-O-IV-1,2-dimethyl-3-hydroxy-4(1H)pyridinone (DHP) with apotransferrin (apoTF) were followed by means of electron paramagnetic resonance spectroscopy (EPR) at room temperature and at liquid nitrogen temperature. Partial ligand displacement between DHP and apoTF was observed, and the reaction was used to determine the binding constant of (VO)-O-IV to apoTF. A value of log K = 14.3 +/- 0.6 was obtained for the binding of the first (VO)-O-IV to apoTF In order to confirm the validity of the binding constant, UV spectroscopy was used to monitor the displacement reaction between (VO)-O-IV-apoTF and nitrilotriacetic acid (NTA). The difference between the binding constants obtained by the two different methods (ca. one log unit) was explained in terms of the possible ternary complex formation between (VO)-O-IV-apoTF and the displacing ligand. The binding constants obtained exhibit a reasonably good agreement with the linear free energy relationship (LFER) estimation.