Characterization of melittin binding to Euplotes octocarinatus centrin

In the presence of 1.0mM Ca(2+), the interaction between Euplotes octocarinatus centrin (EoCen) and melittin (ME) was studied by means of fluorescence spectra. In 0.1M N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and 150mM NaCl at pH 7.4, fluorescence peak of ME was observed at about 353nm indicating that micro-environment of Tryptophan (Trp) residue in ME was hydrophilic. With the addition of 3.2x10(-4)M calcium saturated EoCen (holoEoCen), the peak of ME was blue-shifted to 339nm, which may be resulted from micro-environmental changes of the peptide. At the same time, fluorescence emission of ME was increased significantly suggesting that new complex of ME-holoEoCen was formed under the experimental conditions. Based on the fluorescence titration curves, the 1:1 stoichiometric ratio of holoEoCen to ME was confirmed. In addition, the conditional binding constant of holoEoCen with ME was calculated to be logK(ME-holoEoCen)=6.59+/-0.14.