Adsorption of Euplotes octocarinatus centrin on glassy carbon electrodes as substrates to study europium–protein interactions

Journal of Electroanalytical Chemistry 707 (2013) 102–109

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Adsorption of Euplotes octocarinatus centrin on glassy carbon electrodes as substrates to study europium–protein interactions Zhijiang Rong a,b, Yaqin Zhao a, Bin Liu a, Yanni Tian a, Binsheng Yang a,⇑ a b

Institute of Molecular Science, Key Laboratory of Chemical Biology of Molecular Engineering of Education Ministry, Shanxi University, Taiyuan 030006, PR China School of Environment and Safety, Taiyuan University of Science and Technology, Taiyuan 030024, PR China

a r t i c l e

i n f o

Article history: Received 14 June 2013 Received in revised form 26 August 2013 Accepted 31 August 2013 Available online 7 September 2013 Keywords: Eu3+ Centrin 3=4 FeðCNÞ6 Cyclic voltammetry Ac impedance spectroscopy

a b s t r a c t The adsorption of N-terminal domain of ciliate Euplotes octocarinatus centrin (N-EoCen) onto a glassy carbon (GC) electrode was studied by cyclic voltammetry and electrochemical impedance spectroscopy. The adsorption process obeys Langmuir isotherm adsorption equation. Based on the adsorption, direct immobilization of N-EoCen onto the glassy carbon surface was used for construction of an N-EoCenmodified GC electrode. Then the electrode was used to probe the binding mode of europium ions with N-EoCen. The results show that with the increasing concentration of europium ions, the redox peaks of probe increase gradually. Simultaneously, the peak separation decrease and peak currents of the redox reaction increase. In addition, two binding sites in N-EoCen show no-equiv signal of CV change: the signal is slightly changed by the binding of the first europium ions and largely did by the binding of the second europium ions. It can be attributed to the change of conformation or aggregation of protein after binding of europium ions. It offers a viable model for illustrating the interaction of lanthanides with EoCen. Through the titration curve the process of combination can be explored. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Centrin belongs to the EF-hand calmodulin superfamily of calcium-modulated proteins and play a fundamental role in centrosome duplication and contraction of centrin-based fiber systems [1–3]. It can coordinate four Ca2+ with two high affinity sites in C-terminal and two low affinity sites in N-terminal [4], upon Ca2+ binding, triggered proteins undergo a large conformational change and in turn regulate a vast number of target proteins [5–7]. Ciliate Euplotes Octocarinatus centrin (EoCen) was firstly reported by our laboratory [8]. Lanthanide ions (Ln3+) can react with EoCen occupying Ca2+ binding sites and induce EoCen to undergo conformational changes from closed state to open state, resulting in exposing hydrophobic patches to external environments [9,10]. The binding properties of Ln3+ (La3+, Nd3+, Eu3+, Gd3+, Tb3+, Tm3+, Lu3+) with EoCen were measured by the UV–Vis spectra [11,12], fluorescence spectra [13], circular dichroism spectra (CD) [14], resonance light scattering measurements (RLS) [15] and Cyclic voltammetry (CV) [16]. Self-assembly or namely aggregation, is an important characteristic of centrin in many life process [17]. Previous work proved that lanthanides also lead to the self-assembly of EoCen by spectroscopic measurement. Even though these studies have demonstrated the interaction of Ln3+ with EoCen to

⇑ Corresponding author. Tel.: +86 351 7016358. E-mail address: [email protected] (B. Yang). 1572-6657/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jelechem.2013.08.035

some extent, they are still to the disadvantage of the study of the lanthanides which have no sensitized fluorescence such as Eu3+. Based on our knowledge, few work concerned with using electrochemical methods to investigate the interaction between Ln3+ and EoCen has been reported. The interaction of proteins with solid conducting surfaces has long been an intensely investigated phenomenon [18–21]. Electrochemical methods, such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), reveal the changes in the electrochemical properties of the electrode due to adsorbed protein [22–25]. In all cases it was found that upon adsorption of protein, the electron transfer rates between the electrode surface and electrolyte are severely hindered. This effect was clearly demonstrated by the work performed by Guo et al. [26], using cyclic voltammetry at a gold, platinum and glassy carbon electrode in potassium ferricyanide before and after adsorption of serum albumin. They found that as protein was adsorbed, the formation of the inert protein layer caused a decrease in anodic peak current and an associated oxidation/reduction potential shift to more and less positive values, respectively, resulting in a decrease in the electron 3 transfer in the redox reaction of FeðCNÞ4 ions at the 6 =FeðCNÞ6 electrode surface. Similar studies have been performed, such as haemoglobin, lactalbumin at a platinum electrode [27,28]. In this paper N-EoCen (about 101 residues and a molecular mass of 10 kDa) was gotten by using biological engineering method [8], which is the N-terminal domain of EoCen including the first and

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2. Experimental 2.1. Reagents N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) was purchased from Sigma and used without further purification. All other chemicals are of analytical grade. The Eu3+ stock solutions were prepared by dissolving the appropriate mass of the Eu2O3, in hydrochloric acid, which was then standardized with EDTA in 0.1 M HAc–NaAc buffer at pH 5.5. In the electrochemical measurements, the supporting electrolyte was usually 0.01 M buffers of Hepes containing 0.1 M or 0.02 M KCl. Solutions were prepared with twice distilled water.

2.2. Electrochemical study Electrochemical studies were carried out in a 5 mL cell incorporating three-electrode configuration containing supporting electrolyte, powered by the CHI 660C electrochemical analyzer (CHEN HUA Instrumental Co., Shanghai). A potassium chloride saturated calomel electrode (SCE) and a platinum wire were used as reference and counter electrodes, respectively. A glassy carbon disk electrode (GC) of 3 mm diameter was employed as the working electrode. Prior to each experiment, the GC electrodes were cleaned by polishing with an alumina–water slurry (high-purity Al2O3, particle size 0.3 and 0.05 lm, BDH) and sonicated briefly, followed by thorough rinsing with water. The potential sweep rate used was 50 mV s1 in CV experiments, using a potential window of 0.5 to 0.2 V. As expected, EIS measurement was performed at amplitude of 10 mV and potential of 0.175 V (formal potential of ferro/ferricyanide redox) in the presence of 1.0 mM potassium ferricyanide solution containing 20 mM KCl. The frequency range was from 100 kHz to 0.1 Hz. Impedance data were fitted to appropriate model using the ZSimpWin software (Ametek).

2.3. Protein expression and purification A truncated ciliate EoCen, N-EoCen, including the first and the second EF-hand domain, was obtained using biological engineering methods (see Supporting information). The protein concentration was measured spectrophotometrically at 280 nm using molar extinction coefficients of 4350 M1 cm1 for N-EoCen. The extinction coefficient of N-EoCen was estimated from the Tyr content as described by Pace et al. [29].

2.4. Resonance light scattering Resonance light scattering (RLS) of samples was monitored by fluorescence in quartz cells of 1 cm optical path at 25 °C. The RLS was performed in 0.01 M Hepes at pH 7.4, 0.1 M KCl with a fluorescence spectrometer (F-2500, Hitachi, Japan) using the same excitation and emission wavelengths. Samples were prepared by gradually adding Eu3+ into solution of proteins. An equilibrium time of 5 min was used between each titration.

3. Results and discussion 3.1. The adsorption of N-EoCen onto a glassy carbon electrode Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used for monitoring the effect of exposing the GC electrode to a solution containing N-EoCen on heterogeneous electron transfer. The ferricyanide ions were used as the redox probe. The cyclic voltammograms of a 0.01 M Hepes buffer solution (pH7.4) containing 4.0 lM N-EoCen, 0.1 M KCl supporting electrolyte and 1.0 mM potassium ferricyanide at various exposure times are represented in Fig. 1. The potential sweep rate used was 50 mV s1, using a potential window of 0.5 to 0.2 V. As expected, 3=4 FeðCNÞ6 shows the reversible behavior on a bare glassy carbon electrode (Fig. 1, curve a). In all voltammograms, when the GC electrode is exposed to N-EoCen in the solution, there is a decrease in peak currents and an increase in the peak potential separation over time. In the presence of N-EoCen, the reduction peak potential in the voltammogram was, for example, shifted 96 mV more negative after 280th cycle (130 min) and the corresponding peak current was reduced by 59%, as compared to the reduction peak current at the electrode exposed to the N-EoCen-free solution. A peak current profile for N-EoCen adsorption during 170 min is represented in Fig. 2 (curve b). It was constructed by dividing the backgroundcorrected time-varying cathodic peak current for ferricyanide in the N-EoCen-contained solution (Ip,c(t)) by the cathodic peak current measured at time zero (the cyclic voltammogram recorded immediately after immersion of the GC electrode in N-EoCen-contained solution, (Ip,c(0)). A steady state current was reached at about 130 min later. Therefore, we chose 130 min as protein adsorption time on the GC electrode in this paper. This profile indicates that as the adsorption time increase, the cathodic peak current decrease. The insulating layer blocks the electron transfer between the redox probe and electrode. For comparition, a peak current profile of ferricyanide at bare GC electrodes with 300 scan cycles is shown in Fig. 2 (curve a). There is a small decrease in peak currents upon cycling due to the electrode fouling by polymerized ferricyanide. However, the degree of peak currents decrease in the presence of N-EoCen is greater than that without protein. It is almost negligible for the adsorption effect of potassium ferricyanide compared with protein adsorption. Moreover, considering the isoelectric point of N-EoCen is 4.8, the N-EoCen is negatively charged at pH 7.4. Therefore, the mutually exclusive electrostatic interaction forces are expected between the N-EoCen and ferricynide. So, co-adsorption of protein with ferricyanide is expected to be very small. In order to clarify

a

-10

k -5 I / μA

second EF-hand domain (site I and site II). We constructed N-EoCen film on glassy carbon electrodes by adsorption as interfacial films, with Eu3+ regulating the permeation of FeðCNÞ3=4 to the N-EoCen 6 film to investigate the interaction of Eu3+ with the N-EoCen.

0

5

10 0.5

0.4

0.3

0.2

0.1

0.0

-0.1

-0.2

E vs SCE / V Fig. 1. Selected consecutive cyclic voltammograms of 1.0 mM Fe(CN)63 in 10 mM Hepes, 0.1 M KCl buffer (pH 7.4) in the absence (a) and presence of 4.0 lM N-EoCen (from b to k), using a potential sweep rate of 50 mVs1. The cycle numbers are b: 1; c: 2; d: 3; e: 4; f: 10; g: 20; h: 50; i: 100; j: 200; k: 280.

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1.1

0.7

layer of protein is formed on the electrode. For example, the concentration of N-EoCen added is from 3.9 to 18.3 lM increases the electron-transfer resistance from 52.9 to 101.9 KX. The chargetransfer resistance was very sensitive to the amount of adsorbed protein. Adsorption of N-EoCen onto the electrode surface can be described by the Langmuir isotherm equation [36–40].

0.6

C¼

Ip,c(t) / Ip,c(0)

1.0 0.9

a

0.8

0.5

0

2

4

6

8

10

ð1Þ

Langmuir isothermal equation can be rewritten as:

b

0.4

BADS Cmax C 1 þ BADS C

C 12

3 Time / 10 s Fig. 2. Cathodic current profile (relative time-dependent peak current) for Fe(CN)63/4 in the absence (a)and presence of 4.0 lM N-EoCen (b) using the GC electrode. The data recorded in 1.0 mM Fe(CN)63, 10 mM Hepes buffer (pH 7.4) using a potential sweep rate of 50 mVs1. For curve a: Ip,c(t) denotes the background-corrected time-varying cathodic peak current for Fe(CN)63/4 at a nonzero time, Ip,c(0) indicates the cathodic peak current measured at time zero. For curve b: Ip,c(t) denotes the background-corrected time-varying cathodic peak current for Fe(CN)63/4 in the N-EoCen-contained solution and Ip,c(0) indicates the cathodic peak current measured at time zero (the cyclic voltammogram recorded immediately after immersion of the GC electrode in N-EoCen-contained solution).

the co-adsorption, the following experiments were carried out. The GC electrode is immersed in the a 0.01 M Hepes buffer solution (pH7.4) containing 4.0 lM N-EoCen, 0.1 M KCl supporting electrolyte, but without ferricyanide, and then 280 scan cycle are applied, thereafter 1.0 mM ferricyanide are added, and then CVs of ferricyanide are recorded immediately after solution achieved stable. The value of cathodic peak current recorded was similar to that in Fig. 1, curve k. So, the decreases in peak currents in Fig. 2 (curve b) are mainly attributed to a continuous layer of N-EoCen that had formed on the GC surface, which hinders the electron transfer between the negatively-charged species of ferricyanide and the electrode surface. Impedance spectroscopy is an effective method to probe the interface properties of surface-modified electrodes. We recorded the impedance spectroscopy of a GC electrode in the absense (Fig. 3A) and presense of N-EoCen (Fig. 3B) using 1.0 mM potassium ferricyanide as probe ions at their formal potential in a 10 mM Hepes solution containing 20 mM KCl. The impedance results of Fig. 3A were fitted to the equivalent circuit (in the inset of Fig. 3A). The impedance results of Fig. 3B were also fitted to the equivalent circuit, as shown in Fig. 3D, which is characteristic of an insulating layer over a conducting surface [30–35] and yielded the best fit among several other circuits. To prove the effectiveness of the fitting, fitting results obtained by a non-linear least square procedure are given in Fig. 3A and B (solid lines). In Fig. 3D, the total impedance depends on several parameters such as: Rs, the electrolyte resistance, Rf, an adsorbed protein resistance, CPE1, an adsorbed protein capacitance as a constant phase element. CPE2, a double-layer capacitance as a constant phase element, Rct, charge transfer resistance, Zw, Warburg impedance. Constant phase element, CPE, was introduced instead of pure capacitors in the fitting procedure to obtain good agreement between the simulated and experimental data. From the EIS of Fig. 3A, the impedance show a small semicircle in the high frequency end, and a linear, diffusion controlled behavior (Warburg) on the low frequency side, as expected. The result had shown that its impedance was obviously different with Fig. 3B. In EIS of Fig. 3B, it shows the results of impedance spectrum of the GC electrode at different concentration of N-EoCen. It was clear that the electron-transfer resistance increased with increasing the concentration of N-EoCen, suggesting that an insulating

C

¼

1 C þ BADS Cmax Cmax

ð2Þ

In which C (mol cm3) is the equilibrium concentration of the adsorbate in the bulk solution, C (mol cm2) is the amount of protein adsorbed, i.e., surface concentration, Cmax (mol cm2) is the maximum value of C (saturated surface concentration) and the parameter BADS (cm3 mol1) reflects the affinity of the adsorbate molecules towards adsorption sites. Since Rct is directly proportional to C, substitution of Rct for C, and rearrangement of Eq. (2) gives:

C 1 C ¼ þ Rct BADS Rmax Rmax

ð3Þ

If the Langmuir isotherm is valid for an observed system, a plot of C/Rct versus concentration C should yield a straight line with parameters, Rmax and BADS derived from the slope and intercept, respectively. As can be seen from Fig. 3C, adsorption of N-EoCen is presented, and indeed, the C/Rct versus C dependence is linear, with a correlation coefficient r2 = 0.9952. The parameter BADS = 2.482  105, which reflects the affinity of the absorbate molecules towards adsorption sites at a constant temperature, can be presented by Gomma and Wahdan [41].

BADS ¼

  1 DGADS exp 55:5 RT

ð4Þ

Where R (J mol1 K1) is the gas constant, T (K) the temperature, DGADS (J mol1) the Gibbs energy of adsorption and 55.5 is the molar concentration of the water (mol dm3), which is used as a solvent. Using this equation, the Gibbs energy of adsorption of N-EoCen onto the GC electrode surface in the Hepes buffer solution was calculated at experimental temperature. We obtained the value of the Gibbs energy of adsorption for N-EoCen was 40.86 kJ mol1 in the experimental condition. Such a negative value indicates a spontaneous adsorption of N-EoCen onto the GC electrode. To investigate the probability of desorption of adsorbed N-EoCen from the GC surface, the GC electrode was exposed to the N-EoCen-contained solution in the presence of ferrocyanide as the redox probe and 280 consecutive potential cycles were applied. Then the electrode was rinsed rapidly with distilled water and Hepes and placed immediately in the N-EoCen-free solution (containing ferrocyanide as the redox probe) and consecutive cyclic voltammograms were recorded (data not shown). The peak currents changed only 2.3% after 280 cycles, which indicates that the N-EoCen immobilization at the GC surface is irreversible. 3.2. The interaction between Eu3+ and N-EoCen According to the results represented above, N-EoCen can be immobilized on the GC surface from its solution. The modified GC surface can be used to investigate the interactions of N-EoCen and Eu3+ and to further explore the corresponding conformational changes by Eu3+ binding. The Ln3+ have the similarity to Ca2+ in coordination chemistry, it can compete with Ca2+ at calcium-binding

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A

B

5000

- Z'' / KΩ

a

3000 2000

30 20

1000

10

0

0

0

1000

2000

3000

4000

5000

6000

Z' / Ω

4

CN-EoCen / Rct 10 / μM Ω

−1

C

d

40

4000 - Z'' / Ω

50

0

20

40

60

80

100

Z' / KΩ

1.8 1.6 1.4 1.2 1.0 0.8 0.6 4

6

8

10

12

14

16

CN-EoCen / μΜ Fig. 3. Sensing of different concentration of N-EoCen with the GC electrode. In all experiments the probe solution contains 0.01 M Hepes, 0.02 M KCl, pH 7.4 buffer solution in the presence of 1.0 mM Fe(CN)63, T = 25 °C. (A) Electrochemical impedance spectra of bare GC electrode in the absence of N-EoCen. Inset is Randles model used in analysis of data obtained from A ((Rs) electrolyte resistance, (Cdl) bilayer capacitance, (Rct) charge transfer resistance and (Zw) Wartburg element). (B) Electrochemical impedance spectra of GC electrode in the presence of different concentration of N-EoCen (from a to d): 3.9, 7.7, 11.4, 14.9 lM. (C) The linear relationship of CN-EoCen/Rct vs. CN-EoCen. The data obtained from B. (D) Randles model used in analysis of data obtained from B. Fitting results according to the equivalent circuit are included (solid lines in A and B).

sites [42–44]. In the light of our previous work, the binding of Ln3+ to N-EoCen can induce conformational change of N-EoCen, and contribute to the self-assembly (or to say aggregation) [15]. It is expected that Eu3+ may bind to centrin films on GC electrode surface and then the resulting aggregation (or conformational change) of protein can affect the access of electroactive species of FeðCNÞ3=4 (referred to as the redox probe). Thus the interaction 6 of Eu3+ with N-EoCen can be studied through the electrochemical response of a probe. The GC electrode was exposed to the N-EoCen-contained solution in the presence of ferricyanide and 280 consecutive potential cycles were applied, then N-EoCen films have formed on the surface of the glassy carbon electrode. The response of CV of FeðCNÞ3=4 by Eu3+ binding to N-EoCen film have been investi6 3=4 gated. Fig. 4A and B shows the CVs of FeðCNÞ6 in the 10 mM Hepes buffer solution containing different concentrations of Eu3+, using the bare GC and N-EoCen coated GC electrodes, respectively. For convenient to comparing, divide cathodic peak current (Ip,c) by Ip,c(0) (the cathodic peak current measured at [Eu3+] = 0) and corresponding plots of Ip,c/Ip,c(0) as a function of [Eu3+] are also shown in Fig. 4D (curves a and b). Each peak current was obtained after a CV signal reaching equilibrium. After adding increasing concentration of Eu3+, a slightly decreased peak current has been observed using bare GC electrode. This is maybe due to the electrode fouling by polymerized ferricyanide upon cycling as shown in Fig. 2 (curve a). However, with increasing the concentration of Eu3+, the redox peaks were increased obviously using N-EoCen coated GC electrodes. It implies that the N-EoCen immobilized on GC electrode 3=4 can react with Eu3+. Moreover, in the CV of FeðCNÞ6 at a N-EoCen coated GC electrode in the presence of increasing concentra-

tions of complex Eu3+-EDTA (The Eu3+ has been completely combined by EDTA, [EDTA]/[Eu3+] = 5:1), the peak currents are barely altered (see Fig. 4C and D curve c). This fact points out that the increasing peak currents induced by Eu3+ can attributed to the reaction of N-EoCen with Eu3+. The cathodic peak currents of the redox reaction (shown in Fig. 5A) and peak separations (shown in Fig. 5B) obtained in Fig. 4B as a function of [Eu3+]/[N-EoCen] also have presented. With increasing the concentration of Eu3+, the redox peaks were increasingly obvious. Simultaneously, the peak separation decreased and peak currents of the redox reaction increased. It can be seen from Fig. 5A and B, before 1.0 equiv. of added Eu3+, the peak separation 3=4 decreased weakly and the peak currents of FeðCNÞ6 increased slightly. While with combining the additional 1.0 equiv. of added Eu3+, the peak separation decreased rapidly and the peak currents of FeðCNÞ3=4 increased quickly, then it changes slowly after 2.0 6 3=4 equiv. of added Eu3+. The titration curve shows that FeðCNÞ6 3+ signal varies with the concentration of added Eu due to the combination of Eu3+ to N-EoCen. The first equivalent of added Eu3+ appear to bind quantitatively, but with minimal response, stronger electrochemical response were induced with the addition of another equivalent of Eu3+ and reached a final plateau at the [Eu3+]/ [N-EoCen] ratio up to 2:1. The titration curves revealed two breaks at [Eu3+]/[N-EoCen] = 1.0 and 2.0, confirming that the 2:1 stoichiometric ratio of the Eu2-N-EoCen complex was formed. It can be seen that there are two no-equiv signals of CV change with increasing concentration of Eu3+. Titration curve shows that there are two no-equiv signals of CV change corresponding to the two no-equiv binding site in N-EoCen. CV signal changes with adding Eu3+ imply that Eu3+ disrupts the

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A

-10

-8

8 1

-6

8

-5

-4 I / μA

I / μA

B

1

0

-2 0 2

5

4 10

6 0.4

0.2

0.0

-0.2

0.5

0.4

0.3

0.2

E vs SCE / V

C

0.1

0.0

-0.1

-0.2

E vs SCE / V

D

-6

b

1.6

-2

6

0

1

Ip,c / Ip,c(0)

I / μA

-4 1.4 1.2

2

c

1.0

4

a

6 0.4

0.2

0.0

0.8

-0.2

0

5

10

15

20

25

Eu3+ / μM

E vs SCE / V

Fig. 4. (A) Cyclic voltammograms of 1.0 mM FeðCNÞ36  at bare GC electrode in 0.01 M Hepes, 0.1 M KCl solutions (pH 7.4) containing different concentration of Eu3+(from 1 to 3=4 8): 0, 2.51, 5.59, 7.41, 10.4, 15.1, 20.7, 23.5 lM. (B) Cyclic voltammograms of 1.0 mM FeðCNÞ6 at a N-EoCen coated GC electrode in 0.01 M Hepes, 0.1 M KCl solutions (pH 7.4) containing different concentration of Eu3+ (from 1 to 8): 0, 2.46, 5.48, 7.27, 9.04, 11.9, 17.6, 23.1 lM. (C) Cyclic voltammograms of 1.0 mM FeðCNÞ3=4 at a N-EoCen 6 coated GC electrode in 0.01 M Hepes, 0.1 M KCl solutions (pH 7.4) containing different concentration of Eu3+ (from 1 to 6): 0, 4.30, 8.46, 12.0, 16.4, 20.6 lM. ([EDTA]/ [Eu3+] = 5:1). All the experiments are at scan rate: 50 mV s1. (D) Ip,c/Ip,c(0) as a function of [Eu3+] (Ip,c(0) indicates the cathodic peak current measured at [Eu3+] = 0). Data of curves a, b and c are obtained in A, B, C respectively.

A

B

-7.5

0.30 0.25

-6.5 -6.0

ΔEP / V

Ip,c / μA

-7.0

-5.5 -5.0

0.20 0.15 0.10

-4.5 0

1

2

3

4

Eu3+ / N-EoCen

0.05

0

1

2

3

4

[Eu3+] / [N-EoCen]

Fig. 5. (A) Cathodic peak currents (B) The peak separations of the redox reaction, obtained in Fig. 4B as a function of [Eu3+]/[N-EoCen].

conformation of N-EoCen, then Eu3+-induced conformational changes of N-EoCen were reflected by FeðCNÞ3=4 probe. In light 6 of the decrease of the peak separation and increase of currents, it implies an increasing electron-transfer rate, probe response shows that the electron-transfer rate induced by combination of Eu3+ to site II are bigger than site I. So, in the present work, a novel method has been found which can discriminate two different Eu3+-binding sites of N-EoCen. The adsorption of proteins on carbon electrodes is known to occur without denaturation or loss of biochemical activity in some cases [45–47]. Herein the experiment results just imply

that the biochemical activity of N-EoCen is not affected by the adsorption on GC electrode. However, what kind of conformational change does cause the increase of electron-transfer rate? We further explored it in Sections 3.4 and 3.5. 3.3. Binding properties of Eu3+ to Eu-N-EoCen (Eu3+:N-EoCen = 1:1) The above study shows that the extent of change of CV signal caused by binding of Eu3+ to site I and site II is different. The

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combination of the site II with Eu3+ lead to even more significant current and peak separation changes than site I. In order to further explore the different binding properties between site I and site II, the 1:1 stoichiometric ratio of Eu3+ to N-EoCen (Eu-N-EoCen) was adsorbed on the GC electrode in advance, and its binding properties were studied by FeðCNÞ3=4 probe. Fig. 6A was the cyclic voltammo6 grams changes with titration by Eu3+ to Eu-N-EoCen adsorption layer. As shown in Fig. 6B and C, the peak separation decreased rapidly and the peak currents of FeðCNÞ3=4 increased quickly from 0 to 6 1.0 equiv. of Eu3+. These significant CV changes can be attributed to the binding of Eu3+ to site II. There is no significant change above 1.0 equiv. of Eu3+, indicating all the two binding sites were occupied by Eu3+. The experimental results prove clearly that CV signal change mainly comes from the combination of the site II. It shows the site II play a key role in the course of Eu3+ induced protein conformation changes.

change [13,15]. We have also reported that the aggregation of EoCen induced by lanthanides is much higher than that induced by Ca2+ [15]. Moreover, the N-terminal domain of EoCen plays a critical role in the aggregations [49]. Herein, fluorescent measurements were used to study the protein conformation changes and aggregation properties. RLS is a valuable technique for detecting and characterizing extended aggregates of chromophores, since the aggregations lead to the formation of large fractal structures that exhibit strong RLS signals [50]. A series of fluorescence RLS was conducted at different wavelength between 250 and 600 nm to monitor the aggregation of N-EoCen in 0.01 M Hepes at pH 7.4, containing 0.1 M KCl. Titration curve of RLS versus [Eu3+]/[N-EoCen] was plotted from Fig. S2. As shown in Fig. 7, when a solution of N-EoCen buffer was added to the first 1.0 equiv. Eu3+, RLS increased slightly, and when combined with the additional 1.0 equiv. Eu3+, RLS was enhanced significantly and finally reached to larger amplitude at 2.0 equiv. Eu3+. This RLS experiment shows a similar titration curves to those obtained from electrochemistry experiments (Fig. 5A), which implies that aggregation of N-EoCen induced by Eu3+ play a critical role in electrochemical titration experiments. It indicates that CV signals of probe ion with adding Eu3+ may be arise from protein aggregation triggered by Eu3+. So we speculate that it is due to the aggregation of the N-EoCen on the electrode that make permeability changes for the probe to the electrode surface, which control the electron transfer rate. The increase of electron transfer rate was induced by the increase in permeability of probe ions. According to the results of electron transfer rate for binding with site II is greater than site I, it can be concluded that the aggregation degree of site II is greater than

3.4. Characterization of resonance light scattering in Eu3+-N-EoCen complex Electron transfer in a microstructured film is a process involving the dynamics of several elementary events that overlap and crossinfluence one another. Therefore, this film controls the kinetics of charge transport associated with the observed current. Why can the combination of Eu3+ to N-EoCen cause redox current increase and decrease of the peak separation? A number of cell biology studies on lower eukaryotic cells have demonstrated that centrin plays an important structural role by contributing to the formation of Ca2+-sensitive contractile filaments [48]. Previously we have discovered that aggregation of centrin happened when binding of lanthanides to centrin in addition to conformation

A

-10

h a

I / μA

-5

0

5

10 0.5

0.4

0.3

0.2

0.1

0.0

-0.1

-0.2

E vs SCE / V

B

c

-8.0

250

-7.5

ΔEp / mV

Ip,c / μA

-7.0 -6.5 -6.0

200

150

-5.5

100

-5.0 0

1

2

Eu3+ /

3

Eu-N-EoCen

3=4

4

0

1

2

3

4

Eu3+ / Eu-N-EoCen

Fig. 6. (A) Cyclic voltammograms of 1.0 mM FeðCNÞ6 at an Eu-N-EoCen coated GC electrode in 0.01 M Hepes, 0.1 M KCl solutions (pH 7.4) containing different concentration ratios of Eu3+ and Eu-N-EoCen, from a to g is 0, 0.47, 0.70, 0.93,1.2, 1.7, 2.2, (h) Bare GC electrode without the Eu3+. (B) Cathodic peak currents. (C) The peak separations of the redox reaction, obtained in Fig. 6A as a function of [Eu3+]/[protein].

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Z. Rong et al. / Journal of Electroanalytical Chemistry 707 (2013) 102–109 1200 1000

RLS at 400nm

800 600 400 200 0 0

1

2

3

4

Eu 3+ / N-EoCen

Fig. 7. The titration curve of N-EoCen with the addition of Eu3+, using resonance light scattering value at 400 nm in 0.01 M Hepes, pH 7.4, 0.1 M KCl.

site I. In other words, site II plays a key role in the aggregation process. 3.5. Different adsorption properties of N-EoCen and Eu2-N-EoCen (Eu3+:N-EoCen = 2:1) To confirm the speculation, the following experiment was carried out. N-EoCen protein and Eu2-N-EoCen protein was added gradually into Hepes buffer contains 1.0 mM FeðCNÞ3=4 , respec6 tively. The changes of CV signal of FeðCNÞ3=4 with the addition 6 of protein was observed. The reduction peak currents of FeðCNÞ3=4 versus concentration of adding N-EoCen and Eu2-N-Eo6 Cen obtained from Fig. S1 was shown in Fig. 8. It can be seen that the adsorption behavior of the two kinds of proteins is obviously different. The changes of redox peak currents and the peak separation (Fig. S1) is smaller for Eu2-N-EoCen as compared with N-EoCen at the same concentration of protein. As also can be seen in Fig. 8, the process of protein adsorption at GC electrode can be classified into two phases. The cathodic cur3=4 rent of FeðCNÞ6 present a linear change with the concentration of adding protein. Since the adsorption of N-EoCen obeys Langmuir isotherm adsorption equation, it can be deduced a monolayer adsorption process. The two apparent saturated adsorption concentrations of N-EoCen and Eu2-N-EoCen can be obtained from the point of linear crossing derived from the fitting linear on these two linear adsorption phases. CN-EoCen = 6.86  107 and CEu2-N-EoCen = 2.91  106 M are obtained. The latter concertration

-10

Ip,c / μA

-8 -6 -4 -2 0

10

20

30

40

50

is about four times larger than the former, indicating that the saturated adsorption concentration on GC electrode surface required for Eu2-N-EoCen is greatly higher than that of N-EoCen. Probably the aggregation of Eu2-N-EoCen induced the formation of multimer, and subsequent contraction resulted in less surface area occupied. So it needs more of protein to saturate the same electrode surface. This may be the critical factor that caused the higher saturated adsorption concentration for Eu2-N-EoCen than N-EoCen. It is not surprising that the degree of peak currents decreased by adsorption of Eu2-N-EoCen is significantly less than that caused by N-EoCen at the same concentration of protein added. Fig. 5 is carried out in a low concertration of N-EoCen (4.0 lM). What will happen if a large amount of the N-EoCen is added? Obviously a high amount of protein will be absorbed on the electrode surface. Meantime a lot of free protein will exist in the solution. In this case, aggregation induced by Eu3+ will happen not only between the proteins on the surface of the electrodes, but also between the protein on the electrode surface and those in the solution. Thus at the initial added Eu3+, the protein coverage on the electrode surface will increase due to the continuing adsorption of proteins in the solution. So if the protein concentration in solution is bigger, it should be observed the decrease of the current and the increase of peak separation in the range of [Eu3+]/[N-EoCen] = 0–1. Fig. S3 is the figure of CV signal varied with [Eu3+]/ [N-EoCen]. The total concentration of N–EoCen added is 60.0 lM. It has been proved that the peak current decreases and the peak separation increases in the range of [Eu3+]/[N-EoCen] = 0–1. However, the opposite CV signal is appeared between 1.0 and 2.0. It can be explained by the following reasons. First, few of the free protein will be absorbed. Second, Eu3+ will be combined continuely with N-EoCen and trigger the protein aggregation. Third, attributed to the obvious aggregation of site II, protein aggregation induced in this case will largely reduce the surface coverage of the protein, so that it can be observed that current increase sharply and the decrease of the peak separation obviously. When the concentration ratio is greater than 2.0, CV signal will no longer change because there are only two binding sites existed in N-EoCen. Thus this experiment confirmed our previous speculation (see Fig. S3). Combined with the spectroscopic and electrochemical experiment, we infer that the redox current response with Eu3+ is attributed to the aggregation induced by Eu3+.

4. Conclusion The adsorption of N-EoCen onto GC electrode has been studied by cyclic voltammetry and electrochemical impedance spectroscopy. The results suggest N-EoCen can be absorbed on the GC electrode. The adsorption process obeys Langmuir isotherm adsorption equation. Then the interaction between Eu3+ and N-EoCen has also been investigated by using spectroscopic measurement and electrochemical method. The results show that the two different binding sites in N-EoCen can be discriminated by two no-equiv signal of CV change. It is found that Eu3+-triggered aggregation of N-EoCen play a key role in CV signal changes. The results obtained from both spectroscopic measurement and electrochemical methods are matched well with each other. The present work offers a viable model study for illustrating the interaction of lanthanides with EoCen for understanding molecular mechanism of centrin functions in the cell.

[protein] / μM 3=4

Fig. 8. Plot of cathodic peak currents of FeðCNÞ6 against the concentration of adding [N-EoCen] (d) and [Eu2-N-EoCen ] (j). In all experiments the probe solution contains 0.01 M Hepes, 0.1 M KCl, pH 7.4 buffer solution in the presence of 1.0 mM FeðCNÞ3 6 , T = 25 °C.

Acknowledgements This work was supported by the National Natural Science Foundation of the People’s Republic of China (Grant Nos. 20771068 and

Z. Rong et al. / Journal of Electroanalytical Chemistry 707 (2013) 102–109

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