Spectral studies of interaction between C-terminal domain on Euplotes Octocarinatus centrin and melittin

• 1、Institute of Molecular Science, Shanxi University
• 2、 Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006, China
• 3、Institute of Molecular Science, Shanxi University, Taiyuan 030006, China

Abstract：Centrin is a low molecular mass (20 kDa) protein that belongs to the EF-hand superfamily of calcium-binding proteins. It plays various roles in a number of different cellular functions. The interactions between C-terminal half of Euplotes octocarinatus centrin (C-EoCen) and melittin (ME) were studied by UV spectra and fluorescence spectra in the presence of 1.0 mM Ca2+. In 100 mM N-2-hydroxyethy-lpiperazine-N-2-ethanesulfonic acid (Hepes) and 150 mM NaCl at pH 7.4, UV absorbance of ME at 280 nm was increased significantly in the presence of Ca2+-saturated C-EoCen (holo-C-EoCen) suggesting the interaction of ME with holo-C-EoCen. In addition, the reaction has been measured by fluorescence spectra under the same experimental conditions. In 100 mM Hepes and 150 mM NaCl at pH 7.4, fluorescence emission of ME appeared at about 353 nm. With the addition of holo-C-EoCen (4.1×10-4 M), fluorescence emission of ME was blue-shifted to 335 nm in virtue of micro-environmental changes of the peptide, indicating that new complex of ME/holo-C-EoCen was formed. Meanwhile, fluorescence emission of ME was increased markedly. On the basis of the result of fluorescence titration curves, the 1:1 stoichiometric ratio of holo-C-EoCen to ME was confirmed. And the conditional binding constant of holo-C-EoCen with ME was calculated to be log KME-holo-C-EoCen = 6.18±0.31.

keywords： Centrin Melittin Fluorescence Spectra UV spectra

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