Analysis of Tb3+- and melittin-binding with the C-terminal domain of centrin in Euplotes octocarinatus

Centrin is a low molecular mass (20 KDa) protein that belongs to the EF-hand superfamily. In this work, the interaction between the Tb3+-saturated C-terminal domain of Euplotes octocarinatus centrin (Tb2-C-EoCen) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was investigated using difference UV–vis spectra and the fluorescence spectra methods. In 100 mM N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid (Hepes) at pH 7.4, with the addition of Tb2-C-EoCen, four new peaks were observed at 265 nm, 278 nm, 317 nm and 360 nm by absorptivity compared with blank solution of TNS. At the same time, the reaction could be measured by fluorescence spectra. The fluorescence emission of TNS was shifted from 480 nm to 445 nm in the presence of Tb2-C-EoCen. Meanwhile, its fluorescence intensity was increased markedly. The 1:1 stoichiometric ratio of C-EoCen to TNS was confirmed by fluorescence titration curves. The conditional binding constants of TNS with C-EoCen and Tb2-C-EoCen were calculated to be log K(C-EoCen-TNS)=5.32±0.04 M−1 and log K(Tb2-C-EoCen-TNS)=5.58±0.12 M−1, respectively. In addition, the protein of Tb2-C-EoCen binding with melittin was also studied. Based on the fluorescence titration curves, the 1:1 stoichiometric ratio of Tb2-C-EoCen to melittin was confirmed. And the conditional binding constant of C-EoCen with melittin was calculated to be log Ka′=6.79±0.17 M−1.