Electrochemical Studies Of The Interactions Between Lanthanide Ions And Ciliate Euplotes Octocarinatus Centrin

Centrin belongs to the EF-hand calmodulin superfamily of calcium-modulated proteins. It can combine with natural Ca2+ ions in the biological body, many biological functions of centrin is regulated by the calcium ions. For a long time, our team has been committed to study the interaction between the lanthanides ions and Euplotes octocarinatus centrin(EoCen). Using lanthanide ions rich light, electricity, magnetic properties to study the complexing microenvironment and bonding conditions of calcium ion. Currently the research methods used mainly are UV-Vis spectra, fluorescence, CD spectra and polyacrylamide gel electrophoresis et al. The Ln3+, including La3+, Tb3+, and Lu3+, can compete with Ca2+ at calcium-binding sites. EoCen is composed of four helix-loop-helix domains with two high affinity sites (sites III, IV) for Ln3+ in the C-terminal and two low affinity sites (sites 1,11) in N-terminal. Lanthanide ions play an important role in EoCen self-assembly and the process of combining with its target protein.Electrochemical method is applied to study the interaction between centrin and Ln3+ recently. It shows that a new species was formed when protein P23(one segment of ciliate Euplotes octocarinatuscentrin) was added to a solution of Eu3+. Based on the redox of Eu3+/2+, the interaction between N terminal of EoCen (N-EoCen) and Eu3+ was investigated using the modified electrodes. Results show that Eu3+2-N-EoCen can be reduced by electrochemical method on EPG, agarose, DDAB and MWNT/Nafion/GCE, but the redox peak currents are little on EPG, agarose, DDAB. Whereas, it’s 5 times of redox peak current on MWNT/Nafion/GCE than EPG. MWNT/Nafion film has been proved helpful to promote the redox of Eu23+-N-EoCen. The electrochemical behavior of complex Eu23+-N-EoCen in the MWNTs modified electrode was then investigated in detail, from which a direct, quasi reversible electron transfer of the complex Eu2-N-EoCen had been obtained. We find that a 2:1 complex Eu23+-N-EoCen is formed. According to the Laviron diffusionless-controled equations, a, n, Ks of the complex Eu23+-N-EoCen are 0.53,2.0, and 1.85s-1 respectively. The pH dependence of the formal potential of Eu23+-N-EoCen suggested that two proton was coupled with two electron transfer in the range of pH 5.6～7.4. Eo’-values were independent of pH below pH 5.0. It indicates that Eu3+ is no longer interacted with N-EoCen at lower pH (<5.0). This phenomenon is further confirmed by UV difference spectroscopy. The interaction of C-EoCen with Eu3+ is also explored on EPG. It shows that Eu3+2-C-EoCen can’t be reduced by electrochemical method at the same conditions.In order to prepare the protein modified electrode, the adsorption behavior of EoCen and N(C)-EoCen) at a GC electrode was studied by cyclic voltammetry(CV) and electrochemical impedance spectroscopy(EIS). The adsorption process obeys Langmuir isotherm adsorption equation. It has the different adsorption isotherm for three proteins. The adsorption of N-EoCen onto a GC surface resulted in a bimodal isotherm, while the adsorption of C-EoCen resulted in a single saturation plateau. The adsorption of EoCen resulted in rapid decline with the increase of concentration after reaching a saturation plateau. The different adsorption behavior may come from the different aggregation nature about protein in solution. We obtained the value of the Gibbs energy of adsorption for protein were:N-EoCen was△GADS (EoCen)=-50.04 kJ mol-1; △GADS (N-EoCen)=-40.45 kJ mol-1;△GADS’(N-EoCen)=-33.891J mol-1; [△GADS (C-EoCen)=-41.95 kJ·mol-1. Such a negative value indicates a spontaneous adsorption of proteins onto the GC electrode. The order of adsorption capacity is EoCen> C-EoCen>N-EoCen. It reveals that adsorption of N-EoCen at GC surface depends on ions strength. With increasing the ions strength, the adsorbed amount decreased. It shows that electrostatical forces play the important role in the adsorption of N-EoCen. Electrode potential applied has no effect on the adsorption of protein. In order to clarify the relationship between the adsorption and conformation of protein, the adsorption of Eu23+-N-EoCen, bovine serum albumin (BSA) and CopC are explored. The result shows that the adsorption capacity of Ln3+-N-EoCen reduced compared with N-EoCen. CopC is rich of β-pleated sheet and different from the centrin. Its adsorption ability is also studied. Results show that BSA(rich of a-helix)which has the similar conformation with centrin exhibits stronger affinity for adsorption onto a GC surface, whereas CopC (rich of β-pleated sheet) which has the different conformation with centrin can’t be adsorped. It shows that the conformation of protein greatly affect the adsorption capacity.Based on the adsorption, direct immobilization of N-EoCen onto the glassy carbon surface was used for construction of an N-EoCen-modified GC electrode. Potassium ferricyanide is used as electrochemical probe to study the interaction of Eu3+ with C-EoCen, N-EoCen and EoCen immobilized on a GC surface. The results show that with the increasing concentration of Eu3+, the redox peaks of probe increase gradually and impedance magnitude decreases. From the titration curves of CV and EIS, it shows that EoCen complexes with Eu3+ at 1:4 ratio, whereas N-EoCen and C-EoCen complex at 1:2 ratio. Corresponding to the four binding sites of EoCen, it shows four no-equiv signals of CV or EIS change. The results show that the four different binding sites in EoCen can be discriminated by four no-equiv signal of CV change. Similarly, Ln3+ ion (La3+, Gd3+ Eu3+)/N-EoCen interactions are investigated in same ions strength by EIS. Affinity order is Gd3+≈ Eu3+> La3+>>Ca2+. It is consistent with the order of Ln3+ions potential. A series of fluorescence RLS is conducted. The results obtained from both spectroscopic measurement and electrochemical methods are matched well with each other, but the RLS titration curves can’t discriminate different binding sites in centrin. In order to understand the applicability of this electrochemical method for other protein, with BSA as a model protein, the interaction between BSA and Eu3+ is also investigated by same method. With the increasing concentration of Eu3+, the redox peaks of probe also increase gradually. Ln3+-BSA complex obeys a 1:1 type stoichiometry and binding constant is K=4.26×106. It is clearly different from the result obtained by spectrum method. Therefore, this electrochemical method perhaps can be only applied to the Ln3+-binding which can cause the conformational change of protein.