electrochemical and in situ uv–visible spectroscopic behavior of cytochrome c at a...

Journal of Electroanalytical Chemistry 514 (2001) 67–74

Electrochemical and in situ UV–visible spectroscopic behavior ofcytochrome c at a cardiolipin-modified electrode

Hyun Park, Jang-Su Park, Yoon-Bo Shim *Department of Chemistry, Pusan National Uni�ersity, Keumjung-Gu, Jangjun, Pusan 609-735, South Korea

Received 26 April 2001; received in revised form 16 July 2001; accepted 23 July 2001

Abstract

The interaction of cytochrome c (cyt c) with phospholipids was investigated using electrochemical, in-situ UV–visible, andFTIR spectrophotometric methods, which showed that the electrostatic interaction between cyt c and cardiolipin (CL) gave arapid direct electron transfer. This was studied for the effect of charges of lipids on the redox reaction of cyt c and investigatedfor electrochemical behavior according to the quantity of CL, the accumulation time, pH, temperature, and the stability of theCL layer. The spectroelectrochemical results showed that only the absorption band appearing at 550 nm, which is one of the Qbands, was directly related to the redox reaction of iron ions in cyt c. The kinetic parameters, DO and ko for the electron transferreaction of cyt c on the lipid layer were determined to be 3.09 (�0.02)×10−7 and 2.06 (�0.04)×10−3 cm s−1. © 2001 ElsevierScience B.V. All rights reserved.

Keywords: Cytochrome c ; Lipid; Cardiolipin; Electron transfer reaction of protein

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1. Introduction

The electrochemical processes of proteins havingmetal redox centers have been studied extensively. Ofthese, cytochrome c (cyt c) in particular has been muchstudied because of its documented crystallographicstructure, much physicochemical characterization, andease of purification. However, direct electron transfereven for cyt c has not been observed using bare elec-trodes, since it is difficult to observe the direct electrontransfer process of proteins at a bare electrode due tothe surface adsorption of the proteins [1]. However, themodification of electrode surfaces with electron pro-moters has made it possible to observe direct electrontransfer between protein molecules and an electrode.There have been many reports of the observation ofdirect electron transfer reactions of proteins using pro-moters, such as inorganic [2–4] and organic promoters[5–8] including amino acids [9,10]. Recently, reviewsfor redox reactions of heme-containing metalloproteins[11] and biochemistry at metal � water interfaces [12]

have been reported. There have been a few reports onthe redox reaction of cyt c at a lipid layer [13,14], andthe electrochemical behavior of ferridoxin, an iron–sul-fur protein in bilayer films of cationic lipids on elec-trodes has been reported [15]. Recently, we observedthe direct electron transfer reaction of cyt c on acardiolipin (CL) layer, an anionic lipid layer and stud-ied the detailed behavior of this system.

Cyt c consists of a single polypeptide chain having104 amino acid residues and arranged in a globulartertiary structure [16]. It is a water-soluble heme proteinthat functions as an electron carrier in a biologicalmembrane, which is mainly composed of lipids. It isbelieved that the electron transfer process of cyt c takesplace due to its embedding in the lipid layer withoutdenaturation [16,17]. In our previous works [18,19], theelectrochemical behavior of benzoquinone on an elec-trode covered with a layer of phosphatidylcholine (PC)was reported. This result showed that a lipid layerloaded on an electrode could allow a redox activespecies to immobilize through the ionic interaction andexhibited a peculiar electrochemical reaction in aqueoussolution. Thus, an electrochemical and UV–visiblestudy on the interaction between cyt c and lipid is

* Corresponding author. Fax: +82-51-514-2430.E-mail address: [email protected] (Y.-B. Shim).

0022-0728/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S 0 0 2 2 -0728 (01 )00627 -1

H. Park et al. / Journal of Electroanalytical Chemistry 514 (2001) 67–7468

necessary to understand the direct electron transferreaction in a biological membrane.

Cyclic voltammetry, in-situ UV–visible spectroelec-trochemistry and FTIR spectroscopy were employed toinvestigate the direct electron transfer reaction of cyt c.Several lipids carrying different charges, PC (neutral),phosphatidic acid (PA, −1 charge), and CL (−2charge) were tested as modifiers to observe the directelectron transfer of cyt c. The kinetic parameters forthis reaction were determined by chronocoulometry. Inaddition, we carried out derivative cyclic voltabsorpto-metric (DCVA) experiments to understand which UV–visible absorption bands of cyt c in the CL layer areaffected by applying the redox potential.

2. Experimental

2.1. Materials

CL (from Escherichia coli, Sigma Co.) and choles-terol (CH, Junsei Chemical Co.) were used withoutfurther purification. Cyt c was used after purification asfollows: cyt c from horse heart (type VI, Sigma Chemi-cal Co.) was converted to the fully oxidized form byaddition of excess K3Fe(CN)6 and then purified byion-exchange chromatography on Whatman CM-32,eluted with 0.5 M NaCl+10 mM phosphate buffer atpH 7.0. Eluent containing the purified protein wasconcentrated by ultrafiltration using Amicon YM-3 ul-trafiltration membranes and then dialyzed extensivelyto remove phosphate [20]. PC was extracted from eggyolk and purified by silica gel (silica gel 60, Merck Co.)column chromatography [21]. PA was prepared fromPC by hydrolysis in the presence of cabbage phospholi-pase D [22,23]. The purity of PA was confirmed bysilica gel thin layer chromatography (TLC, Merck Co.).Distilled water (18 M� cm−1) was obtained from aMilli-Q system. The buffer solutions were preparedusing NaH2PO4+Na2HPO4 and citric acid+sodiumcitrate mixtures. All other reagents used were of thebest commercial quality available.

2.2. Electrodes and apparatus

Before each experiment, a glassy carbon (GC-30S,Tokay Carbon Co.) electrode (GCE) was polished me-chanically with 0.1 �m alumina powder to a mirrorfinish and sonicated for 2 min, then rinsed with ethanol.All lipids were dissolved in a chloroform solution toobtain an adequate concentration (10 mg ml−1). Tomodify the electrode surface, a 3.0 �l aliquot of thesolution was dropped onto the surface of the GCE. Thesolvent was then evaporated by slowly rotating theelectrode (200 rpm) to give homogeneity of the castlipid layer. Applied electrode potentials were measured

with respect to a Ag � AgCl � KClsat electrode, and thecounter electrode was a Pt wire. The temperature of themeasuring solution was controlled at 25�0.1 °C.

A Pine Instrument Co. model AFRDE4 bipoten-tiostat and a PAR model 273 potentiostat/galvanostatwere used to record cyclic voltammograms (CVs) andto control the electrode potentials during the spec-troelectrochemical measurements. In-situ UV–visiblespectroscopic measurements were made with an OceanOptics Co. Model S 1000 spectrograph in the reflec-tance mode coupled with a CCD array detector, axenon lamp, and an optical fiber probe. The bifurcatedfiber optical beam probe was located on the window ofthe quartz electrochemical cell, in which the workingelectrode faced the window of the cell [18]. IR spectrawere obtained with a Mattson Polaris FTIRspectrometer.

3. Results and discussion

3.1. Electrochemistry of cyt c on the CL-modifiedelectrode

The spin coating method was employed to form athin film of CL, and this was compared with a CL filmcoated by the Langmuir–Blodgett (LB) method toconfirm the physical state of the lipid layer. CVs of5.0×10−5 M cyt c (pH 7.0) were recorded at a scanrate of 50 mV s−1 using CL coated electrodes preparedby both methods. The CV results showed no differencefor the CL film formed by the LB method and thatformed by a spin-coating method. This showed that theCL layer formed by the spin-coating method had thesame surface condition with respect to that formed bythe LB method, while it was not an exact monolayer ofCL. CH was mixed with CL for long-time voltammetricmeasurements to enhance the physical stability of thecoated lipid membrane. The thicknesses of the spin-coated layers for both CL and CL–CH were deter-mined as about 30 nm using scanning electronmicroscopy (SEM). SEM pictures show homogeneousmorphology of the lipid layers (not shown). Thus, weused the spin coating method to modify the electrodewith lipids.

The effect of the ionic charges of the lipids on theelectrochemistry was investigated for cyt c having apositive net charge (+7) [24]. PC (neutral), PA (charge;−1), and CL (charge; −2) (Fig. 1) were selected as themodifiers based on the charge of each lipid, one ofwhich brought about the electrostatic interaction theneffected the charge transfer reaction of cyt c at theelectrode surface. The redox peak of 5.0×10−5 M cytc in phosphate buffer (pH 7.0) solution was not ob-served using a bare GCE, or PC- and PA-modifiedGCEs (Fig. 2a). However, the redox peak was observed

H. Park et al. / Journal of Electroanalytical Chemistry 514 (2001) 67–74 69

on the CL-modified electrode (Fig. 2b). The redox peakof cyt c due to the direct charge transfer appeared at+0.090/−0.002 V (�Ep=92 mV) versus Ag � AgCl.The absorbed amount of cyt c on the CL layer was2.7×10−11 mol cm−2, which was calculated from theCV data using the equation of

Ip=n2F2

4RT�A�*O

Fig. 3. CVs recorded in a 5.0×10−5 M cyt c+phosphate buffer (pH7.0) solution with a scan rate of 50 mV s−1.

Fig. 1. Chemical structure of lipids: (a) phosphatidylcholine (PC), (b)PA, and (C) CL.

(where Ip is the peak current, n the number of electrons,F the Faraday constant, R the gas constant, T temper-ature, � the scan rate, A the area of the electrode, and� the absorbed amount of the redox compound). Thisbehavior can be explained by the fact that the presenceof sufficient electrostatic interaction between the nega-tive charges of CL and the positive charges of the lysineresidues around the heme site of cyt c allows the directelectron transfer on the CL modified electrode to takeplace.

Fig. 3 shows the dependence of the CV peak currentof cyt c, recorded for the CL-coated GCE in cytc-containing buffer solution during continuous poten-tial cycling between 0.15 and −0.15 V, on the reactiontime. The redox peak currents of cyt c at a CL-coatedelectrode increased as the number of potential cyclesincreased, and then the peak current reached a steadystate over a certain number of cycles �20. When theCV was recorded after transfer of the CL-modifiedelectrode, fully absorbed with cyt c, into pure phos-phate buffer (pH 7.0), it showed the same pattern as theCV recorded in the cyt c solution during potentialcycling. These results show that the CL-coated elec-trode captured cyt c from the solution by the electro-static interaction between cyt c and the CL layer. Thecaptured cyt c on the CL layer from the bulk solutionis stable and has the same electrochemical activitycompared to that in the solution containing cyt c.

The plots of peak current versus the scan rate showthat the current is directly proportional to the scan ratefrom 10 to 50 mV s−1. However, the peak currentswere proportional to the square root of the scan ratefrom 70 to 200 mV s−1 (not shown in the figure). Thisbehavior is very similar to the result for p-benzo-quinone on the PC layer [18]. In this case, a finitediffusion model is operative during the lower scan rateof below 50 mV s−1, whereas a semi-infinite modelapplies at the higher scan rates above 70 mV s−1. Thisis similar to charge transport via physical diffusion of

Fig. 2. CVs of cyt c (5.0×10−5 M) in phosphate buffer (pH 7.0) on(a) a bare glassy carbon and (b) a CL modified electrode. The scanrate was 50 mV s−1.


the electroactive reactants incorporated in the films,which are attached to the functional groups present inthe polymer films [25]. From these experiments, themechanism of the heterogeneous electron transfer be-tween cyt c and a lipid-modified electrode may beexplained as follows. The hydrophilic sites of a lipid,especially the negative charged phosphate group, inter-act with lysine–NH3

+ groups of cyt c through hydrogenbonding or/and electrostatic interaction. This assumesthat the interaction between cyt c and lipids causes theexposure of the heme group to the outside of theprotein on the electrode or provides a route for theelectron transfer. Direct electron transfer on the CLcoated electrode occurs and the hydrophobic alkylchain of the lipid may prevent the denaturation of cyt c.We will discuss this interaction in detail in Section 3.4.

3.2. Parameters affecting the stability of the CL layer

Some parameters that affect the stability of the CLlayer on the electrode were investigated. Each measure-ment took more than 15 min to accumulate cyt ccompletely onto the CL layer. However, the CL layerwas desorbed, particularly at pH greater than 8. Amicroscope and SEM confirmed this before and afterthe experiments. This is mainly due to the lower stabil-ity of the lipid layer in alkaline solution. In addition,the CV recorded for cyt c adsorbed on the CL coatedelectrode with consecutive cycling in a blank electrolytesolution shows a gradual decrease of the redox peakheight of cyt c. Over three cycles, the peak height wasdecreased by about 30%. This indicates that the cyt ccoated CL layer was unstable in a long time experi-ment. The CL layer was coated on the CH under-layerto stabilize the CL layer [26] because the CL onlycoated layer did not remain stable on the electrodesurface during a long time experiment. The CV patternsof cyt c before and after formation of the under-layer ofCH were both the same and the amount of cyt cabsorbed on the CL layer using the CH under-layer was2.1×10−11 mol cm−2, which was a similar quantity tothat adsorbed on the CL only layer. The CH under-layer was electrochemically inactive and gave a highermechanical stability of the modified electrode surface.No redox peaks were observed for the CL layer alone.

3.2.1. Optimum quantity of CLThe optimum quantity of CL for the electrode mod-

ification was determined to obtain the most reversibleredox wave in this experiment. Fig. 4 shows the plot ofpeak currents (�: Ipa and �: Ipc) according to thequantity of CL on the GC electrode. The CVs wererecorded in a blank phosphate buffer solution (pH 7.0,30 °C), followed by 15 min deposition in a 5.0×10−5

M cyt c+phosphate buffer solution (pH 7.0, 30 °C). A5.0 �l CL+chloroform solution gave the best results inthe cyclic voltammetry, and an increased quantity ofCL caused the peak current to decrease. This wasconsistent with the results that used a cyt c layer coatedby the LB method. The reversibility of the redox reac-tion of cyt c decreased as the CL layer formed by theLB method became a multilayer. We assume that thisoccurs for two reasons, (i) the overspreading of thehead groups of CL and (ii) the increase of the electroderesistance due to the CL coated over CH.

3.2.2. The effect of the accumulation timeTo elucidate the electrostatic interaction between the

CL layer and cyt c, CVs recorded for cyt c adsorbed onthe CL layer were investigated according to the accu-mulation time and the consecutive potential cycling.Fig. 5 shows the plot of the peak current according tothe accumulation time without applying any potential.

Fig. 4. The plot of peak currents (�: Ipa and �: Ipc) according to thequantity of CL coated on the GC electrode.

Fig. 5. The plot of the variation of the peak current (�: Ipa and �:Ipc) with the accumulation time without applying a potential.


Fig. 6. The pH dependence of the redox peak of cyt c on a CL coatedelectrode. (a) 2.3, (b) 4.6, (c) 7.0, (d) 8.0.

shows CVs recorded for adsorbed cyt c on the CH–CLcoated GC electrode in blank solutions of (a) pH 2.3,(b) pH 4.6, (c) pH 7.0, and (d) pH 8.0, after accumula-tion for 10 min in 5.0×10−5 M cyt c+buffer at30 °C. A cathodic peak appeared at −0.272 V at pH2.3, which shifted to −0.340 V at pH 4.6. On the otherhand, one pair of redox peaks was observed at +0.005to −0.075 V at pH 7.0. The CV recorded at pH 8.0reveals the same pattern as that at pH 7.0, but the peakheight was decreased to 10% of that obtained at pH 7.0.However, a part of the CL layer was desorbed at pH 8.This was confirmed before and after measurementsusing a microscope and SEM. This is due to the poorstability of the lipid layer in an alkaline solution as wasmentioned previously. The irreversibility of the redoxreaction of the adsorbed cyt c at a low pH may becaused by the weak interaction between the two speciesdue to the neutralized negative charge of CL by proto-nation of cyt c in the more acidic solution (�pH 6).Thus, direct electron transfer between the two specieswould not be effective in acidic media.

3.2.4. The effect of temperature on the redox beha�iorof cyt c

The transition temperature can be estimated from thetemperature dependence of the redox current of cyt c.To study the influence of the temperature on the redoxreaction of cyt c, CVs were recorded for a CH–CLcoated GC electrode in a blank solution (pH 7.0),followed by 15 min accumulation of cyt c in a 5.0×10−5 M cyt c+phosphate buffer (pH 7.0) solution.The plot of the peak current versus the temperatureshows an S shaped curve having an inflexion pointaround 25 °C, at which the current jump occurs. Thedata are shown in Table 1. This may arise from theelevated mobility of the charged ions in the lipid layerdue to the high fluidity of the CL layer around 25 °C.It is well known that the fluidity of most lipid mem-branes is at a maximum at about 40 °C [27]. The phasetransition temperature of CL is 23–28 °C, higher thanthat of phosphatidylglycerol, which has the samealiphatic chain compared with CL in its hydrophobictail. In addition, this could be explained by an unfold-ing of cyt c, since it is known that the formal potentialbecomes more negative as the exposure of the hemegroup to the solution increases [28,29]. Thus, it may be

The CV was recorded for the CH–CL coated electrodein a blank phosphate buffer solution (pH 7.0, 30 °C),followed by the accumulation of cyt c in a 5.0×10−5

M cyt c+phosphate buffer solution (pH 7.0, 30 °C).The maximum current was attained at an accumulationtime of 15 min and the maximum adsorbed amount ofcyt c was 7.74×10−11 mol cm−2. Then, the peakcurrents gradually decrease with increasing time andthe accumulation over 40 min produced a steep de-crease of the redox peak height to 30% of that for 15min. This might be due to the fact that a long timeexposure of the electrode in the solution led to the lossof the surplus adsorbed CL from the electrode surfaceinto the bulk solution.

3.2.3. The effect of pH on the charge transfer reactionThe redox behavior of cyt c captured on the lipid

layer was investigated in various pH media. Fig. 6

Table 1Peak potentials and currents in CVs recorded for cyt c on CL coated electrodes at scan rate of 100 mV s−1 according to the variation of thereaction temperature

15105Temp./°C 20 3525 4030

Ipa/Ipc/�A 0.73/0.730.50/0.500.45/0.440.39/0.38 0.83/0.830.81/0.820.38/0.37 0.80/0.80+0.097/+0.108/+0.113/Epa/Epc/V +0.094/ +0.070/ +0.077/ +0.081/+0.095/

−0.010 −0.015−0.005−0.006 −0.013−0.012 −0.013−0.015128 120 104 9490�Ep/mV 85103 100


Fig. 7. In-situ UV–visible spectra obtained in a 5.0×10−5 M cytc+phosphate buffer solution (pH 7.0) with CL-coated and bare(inset) GC electrodes: at (a) 200, (b) 100, (c) 0, (d) −100, and (e)−200 mV vs. Ag � AgCl.

of cyt c at the CL modified electrode was evaluatedfrom Eq. (2) for a quasi-reversible electron transferreaction [32].

�=��1/2=(DO/DR)�/2ko

[DO��(nF/RT)]1/2 (2)

where � is an equivalent parameter, DO and DR arediffusion coefficients which are assumed to be approxi-mately the same, � is the scan rate, R is the gasconstant, and T is the thermodynamic temperature [28].By using Eq. (2), the heterogeneous rate constant (ko)of cyt c with a CL-modified GC electrode can becalculated by the plot of � with respect to 1/�1/2 (for thequasi-reversible reaction) in a 5.0×10−5 M cyt c+phosphate buffer solution (0.1 M, pH 7.0). The value ofko was determined to be 2.06 (�0.04)×10−3 cm s−1.This is similar to the reported value, which was 2.0×10−3 cm s−1 for cyt c552 on the 4,4�-dipyridylmodified micro-carbon electrode [33].

3.4. In-situ UV–�isible spectrometry and IR spectra

Fig. 7 shows in-situ UV–visible spectra obtained in a5.0×10−5 M cyt c+phosphate buffer solution (pH7.0) with CL-coated and bare (inset) GC electrodes: theapplied potentials were (a) 200, (b) 100, (c) 0, (d)−100, and (e) −200 mV versus Ag � AgCl. When thenegative potential was applied to the cell, the intensitiesof the absorption bands at 410, 520 and 550 nm wereincreased.

As a result of a strong configuration interactionbetween both transitions, the absorption spectrum dis-plays one strong absorption band, the Soret band (at�400 nm), and a weaker absorption band, the �/�band (at �550 to 520 nm). It is well known thatferricytochrome c exhibits two major bands, a Soretband and a � band appearing at 520 nm withoutapplying any reduction potentials. On the other hand,ferrocytochrome c, obtained by the electrochemical re-duction, exhibits three bands, the Soret band shifted to416 nm, a new � band appearing at 550 nm, and a �band at 520 nm [32]. UV–visible adsorption bands ofheme proteins are dominated by two ��* electronictransitions as shown in Scheme 1 [34]. The bandscorresponding to transitions 3, 4, and 5 have beenidentified in spectra of high-spin ferric systems near 600nm, and in spectra of low-spin ferric systems between1200 and 1500 nm. The situation with ferrous systems isless clear. In low-spin systems transitions 4 and 5 (and8 and 9) cannot occur, because the dxz/dyz orbital paircontains four electrons, but in high-spin systems theycan occur, along with transition 3. However, unequivo-cal band assignments have not been reported for fer-rous proteins.

To assign the absorption bands of cyt c on the CLlayer in detail, the derivative absorption curves [18]

suggested that the current jump takes place by theelevated fluidity of CL and unfolding of cyt c.

3.3. Kinetic parameters for the electron transferreaction between cyt c and a CL layer

To determine the diffusion coefficient, we plotted thecharge with respect to t1/2 (from chronocoulometricdata obtained in a 5.0×10−5 M cyt c+0.1 M phos-phate buffer solution (pH 7.0)). Before the chronocou-lometry for the cyt c, DO of K3Fe(CN)6 chosen as thereference material was determined with a Pt electrode ina 1.0 M KCl solution and showed good agreement withthe value in the literature [30]. The potential wasstepped from 200 to −200 mV versus Ag � AgCl. Wecalculated the diffusion coefficient (DO) using Eq. (1),

Q=2nFADO

1/2c*Ot1/2

�1/2 +Qd,l+nFA�O (1)

where F is Faraday’s constant, A is the area of theelectrode (0.139 cm2), cO* is the concentration of thebulk solution (5.0×10−5 M), and n is the number ofelectrons involved in the electrochemical reaction. Thecalculated diffusion coefficient was found to be 3.09(�0.02)×10−7 cm2 s−1 from the equation of Q/c=9.56×10−8× t1/2+1.08×10−8. This is similar to thevalue given in a previous report [31], which was 5×10−7 cm2 s−1 for cyt c on tin-doped indium oxide andtin oxide electrodes.

Although the peak separation of the redox wave was56 mV at a scan rate of 20 mV s−1, the peak separationbroadened gradually to more than 60 mV at scan ratesover 40 mV s−1, indicating that the process was notfully reversible under our experimental conditions.Thus, the exchange rate constant ko for the oxidation


Scheme 1. Schematic diagram of the electron promotion transitionsresponsible for the optical absorbance of ferricytochromes [34].

peaks of the CV. This indicates that the redox reactionof the metal ion in the heme should contribute only tothe band at 550 nm. The absorption bands at 520 and550 nm are due to the �–�* transition of the porphyrinring inferred from the intensities of these peaks. If boththe � and � bands were due to the electron transferreaction of cyt c, both should show the agreement withthe DCVA curve. However, the DCVA curve for onlyone peak at 550 nm matched with the CV, showing thedirect electron transfer by the heme. Thus, we con-cluded that the �–�* transition of heme (1 and 2transitions in the energy diagram of Scheme 1) and thecharge transfer bands of Fe (dxz, dzy)−�*(eg) or Fe(dxz, dzy)–�* (the axial ligand) transitions (10 and 11transition in the energy diagram) are overlapped andappear at 550 nm. Another possible interpretation ofthe band at 550 nm is that it is due to the electrontransition from the d orbital of iron ions in the heme,i.e. one of the iron electrons could fall into a2u(�),leaving a hole in the metal to be filled by the electron ineg(�*) [35].

In addition, to confirm the interaction sites of cyt cand CL, IR spectra were obtained for the mixture ofcyt c and CL as shown in Table 2. The splitting of aband corresponding to the amide II spectral region inthe IR spectrum of cyt c and the shift of the stretchingbands of the �PO2

− group in CL show that lysinegroups of cyt c interact with �PO2

− groups of CL. Inthe case of the cyt c+CL mixture, bands at 1545.18and 1555.79 cm−1 appeared in the amide II spectralregion of cyt c. An absorption band at 1545.18 cm−1

arising from the amide II spectral region was split intotwo bands at 1545.18 and 1555.79 cm−1 when cyt c wasadsorbed on the CL layer. This band is unique appear-ing only for horse heart cyt c, which arises from thedeformation of �NH3

+ in lysine residues [36]. Twoasymmetric and symmetric stretching bands of �PO2

−

appeared at 1231.33 and 1103.35 cm−1 in pure CL andwere shifted to 1239.03 and 1099.23 cm−1 when cyt cwas dropped on the CL layer. These results mean thatlysine groups in cyt c interact with �PO2

− in the CLlayer through electrostatic interaction.

Fig. 8. The CV (dashed line) and DCVA curve (solid line) of5.0×10−5 M cyt c (pH 7.0) with CL-coated GC electrodes at 550nm. The scan rate was 2 mV s−1.

were recorded for the cyt c on the modified electrodewith the lipid in the phosphate buffer medium. Fig. 8shows the CV (dashed line) along with the derivativeabsorbance (dA/dt) curve recorded at 550 nm (solidline). Only the curve at 550 nm agrees with the redox

Table 2FTIR frequencies of cyt c, CL, and cyt c-CL species

Cyt c-CL, �/cm−1CL, �/cm−1Cyt c, �/cm−1

3425.923405.19�NH �NH or�OH3299.61�NH or �OH�CH 2961.64 �CH 2922.11 �CH 2925.97

�C�CO� 1740.441653.66 �C�CO��CO�N� 1739.48O�C� O�C��CO�– 1661.37– �CO� 1658.48

1555.79�NH3+–�NH3

+ –1545.181545.18

– 1239.52– �PO22−1231.33�PO2

2−

1050.321062.59


4. Conclusion

The direct electron transfer reaction of cyt c wasobserved by the incorporation of cyt c into the CL layerthrough the interaction with PO2− groups of CL andlysine groups of cyt c. The diffusion coefficient and theheterogeneous rate constant of the cyt c on the CLlayer were determined to be 3.09 (�0.02)×10−7 and2.06 (�0.04)×10−3 cm s−1, respectively. The modifi-cation of the electrode with a CL layer led to a rapiddirect charge transfer of cyt c through the electrostaticinteraction between CL and cyt c which was confirmedby UV–visible and IR spectroscopic techniques. Thespectroelectrochemical results showed that only the ab-sorption band at 550 nm, one of Q bands, was relateddirectly to the redox reaction of the heme iron in cyt c.

Acknowledgements

This work was supported financially by the Centerfor Integrated Molecular Systems through the KoreaScience and Engineering Foundation and partially by agrant of the Korea Health 21 R&D Project, Ministry ofHealth and Welfare, Korea (HMP-99-E-11-0004).

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