Investigation on the binding of TNS to centrin, an EF-hand protein.

The interaction between 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and ciliate Euplotes Octocarinatus centrin (Cen) has been studied by fluorescence spectroscopy. The binding constants of TNS with Cen were measured at different temperature in the 0.01M Hepes, pH 7.4. The binding process is exothermic and involves a positive entropy change. The negative value of enthalpy predominately contributes to the negative free energy of binding between TNS and Cen. The salt (KCl) increases the association constant of TNS and Cen. These results and resonance light scattering experiment suggest that the binding force between TNS and Cen is hydrophobic. The distance (r) between TNS and tryptophan of mutant G115W, which sheds more insight into the binding of TNS to Cen, was determined as 4.85nm based on Förster non-radiative energy transfer theory.