An HPLC method associated with a thermodynamic analysis to compare the binding of TRAIL and its nanovectorized form to death receptors DR4 and DR5 and their relationship to cytotoxicity

TRAIL is a member of the TNF family of cytokines which induces apoptosis of cancer cells via its binding to its cognate receptors, DR5 a high affinity site and DR4 a site of low affinity. Our working group has recently demonstrated that nanovectorization of TRAIL with single wall carbon nanotubes (abbreviated NPT) enhanced TRAIL affinity to the high affinity site DR5 and increased pro apoptotic potential in different human tumor cell lines. In this paper, the DR4 low affinity site was immobilized on a chromatographic support and the effect of temperature on a wide temperature range 1 degrees C-50 degrees C was studied to calculate the thermodynamic parameters of the binding of TRAIL and NPT to DR4 and DR5 receptors. For the first time the heat capacity changes for the different binding processes were determined. At a physiological pH (7.4) the heat capacity changes for the binding of NPT to DR4 and DR5 were respectively equal to -0.91 kJ/mol K and -0.28 kJ/mol K and those obtained for the binding of TRAIL to DR4 and DR5 were respectively equal to -1.54 kJ/mol K and -1.05 kJ/mol K. By the use of differential scanning calorimetry (DSC), a phase transition (similar to 12 degrees C for DR5, similar to 4 degrees C for DR4) between a disordered (low temperature) and an ordered (high temperature) solid like state visualized in the receptor structure confirmed the temperature dependence of binding affinity enthalpy Delta H for soluble TRAIL and its nanovectorized form to its cognate receptors. In the low temperature domain, the positive Delta H values contribute non-favourably to the free energy of binding, TRAIL and NPT described similar affinities for DR4 and DR5. For the high temperature domain, negative Delta H values indicated that van der Waals interactions and hydrogen bonding are engaged favourably at the ligand - receptor interface. Above 30 degrees C, their rank-ordered affinities were thus strongly different in the sequence: TRAIL(DR4)