Journal ofElectroanalytical

Chemistry

Journal of Electroanalytical Chemistry 568 (2004) 143–149

www.elsevier.com/locate/jelechem

An electrochemical study on bovine erythrocyte superoxide dismutase– the novel electrochemical behaviors on mercury electrodes

Wen Qian, Qin-Hui Luo *, Zhi-Lin Wang, Meng-Chang Shen

Coordination Chemistry Institute, State Key Laboratory of Coordination Chemistry, Nanjing University, 22 Hankou Road, Nanjing 210093, PR China

Received 13 August 2003; accepted 24 January 2004

Available online 20 March 2004

Abstract

The electrochemical behavior of bovine erythrocyte superoxide dismutase (BESOD) on a hanging or dropping mercury electrode

was studied by cyclic voltammetry (CV) and normal pulse polarography (NPP). Two defined peaks in CV or in NPP diagrams were

attributed to the peaks of copper(II) and zinc(II), respectively. The electrode processes were irreversible and possessed an adsorptive

characteristic. The adsorptionofBESODwas estimatedbydouble potential-step chronocoulometry. The average area of eachmolecule

of absorbed BESOD was obtained, which is in good agreement with that determined by X-ray structure analysis. The diffusion co-

efficients were determined byNPP and double potential-step chronocoulometry, being in agreement with each other. The experimental

results confirm that in the absence of a promotor or mediator, mercury electrodes are feasible for the investigation of BESOD.

� 2004 Elsevier B.V. All rights reserved.

Keywords: Superoxide dismutase; Electrochemical behavior; Adsorption; Mercury electrodes

1. Introduction

The main role of superoxide dismutase (SOD) iscatalyzing the dismutation of superoxide anion [1] and

protecting cells from the toxic effects of superoxide an-

ion [2]. Cu2Zn2SOD is composed of two identical sub-

units, each containing one copper(II) and one zinc(II)

ion, which are connected by a histidine imidazolate

bridge [3]. The catalytic reaction occurs at the site of

copper, and zinc plays the role of stabilizing the protein

structure [4]. The catalytic activity of Cu2Zn2SOD isrelated to its redox properties [5–7], therefore many

scholars have studied its electrochemical behavior [8–

13]. Valentine and coworkers [12] have studied the

temperature dependence of the reduction potential of

Cu2Zn2SOD by spectrochemistry. Afterwards the redox

potentials of Cu2Zn2SOD and its mutants were also

studied using a gold electrode [13]. In their systems,

promotors or mediators were used in order to modify

* Corresponding author. Tel.: +86-25-359-4030; fax: +86-25-5331-

7761.

E-mail address: qhluo@jlonline.com (Q.-H. Luo).

0022-0728/$ - see front matter � 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jelechem.2004.01.014

the surface of the electrodes in order to enhance the

rates of electron transfer and the reversibility of the

electrode reactions [14]. A large number of papers andseveral reviews on the adsorption and reduction of

proteins on mercury electrodes have been reported

[15,16]. However, only a few papers concerning the

electrochemical behavior of Cu2Zn2SOD on mercury

electrodes have appeared to date [17]. Recently, we re-

ported briefly the redox behavior of porcine superoxide

dismutase (PESOD) and of its reconstituted products on

hanging mercury electrodes [18] as well as that ofCo2Co2SOD and Cu2Co2SOD on a pyrolytic graphite

electrode, all in the absence of promotors or mediators

[19]. In this paper, the electrochemical behavior of bo-

vine erythrocyte superoxide dismutase (BESOD) on a

hanging mercury electrode (HME), a dropping mercury

electrode (DME) and a mercury pool electrode, in the

absence of promotors or mediators was researched in

detail by cyclic voltammetry (CV), normal pulse polar-ography (NPP), and double potential-step chronocoul-

ometry. The experimental results showed that BESOD

was adsorbed on the surfaces of the electrodes and

that the electrode processes possessed adsorptive

144 W. Qian et al. / Journal of Electroanalytical Chemistry 568 (2004) 143–149

characteristics. In addition, the average molecular area

of BESOD was obtained from the adsorptive amounts

by double potential-step chronocoulometry and the

protonation constant of the imidazolate bridge in re-

duced SOD was determined by CV. These values are ingood agreement with those obtained by X-ray structure

analysis and by using a Au electrode, respectively [13].

They confirm that this direct electrochemical method on

the HME or DME is feasible for the investigation of

Cu2Zn2SOD.

2. Experimental

2.1. Materials

Bovine erythrocyte superoxide dismutase was pre-

pared from bovine blood by the methods of previous

authors [5]. The raw extract of BESOD was purified on

DEAE-cellulose 32 and Sephadex G50, successively.

The purified BESOD had only one homogeneous andclear band in polyacrylamide gel electrophoresis. The

molar mass of BESOD was measured as 32 800 by SDS-

polyacrylamide gel electrophoresis and its Cu and Zn

contents were measured by inductively coupled plasma

spectrometry (ICP) as 0.398% and 0.402%, respectively,

which were consistent with the calculated values of

0.387% and 0.399%. The specific activity was measured

by the xanthine oxidase method as 6000 U mg�1. Theparameters of EPR and peak values of UV spectra were

in agreement with those in previous reports [20]. Re-

constitution of protein was carried out by routine

methods [21]. The apoprotein of Cu2Zn2SOD, namely,

E2E2SOD (E2 denotes two cavities in SOD, E2E2SOD

denotes that both two copper and two zinc ions were

removed) was prepared [22] by dialysis of a solution of

Cu2Zn2SOD against an acetate buffer solution con-taining EDTA at pH 3.8. After the removal of metal

salts and EDTA, the residual contents of Cu and Zn in

E2E2SOD were measured as 0.05% and 0.06%, respec-

tively, by ICP. The Zn-free derivative Cu2E2SOD was

obtained by exhaustive dialysis of Cu2Zn2SOD against a

phosphate buffer solution at pH 4.8 [21]. The contents of

Cu and Zn in Cu2E2SOD were 0.378% and 0.05%, re-

spectively (calculated Cu content was 0.399%), and thepercentage of reconstitution for copper was 93%. The

Cu-free derivative E2Zn2SOD was prepared by adding

diethyldithiocarbamate (DDC) to a solution of

Cu2Zn2SOD with a Cu2Zn2SOD/DDC ratio of 2:1.

After the mixture was stirred for 24 h, the solution was

dialyzed exhaustively against a phosphate buffer solu-

tion containing 0.1 mol dm�3 NaCl at pH 7.4 to removal

the Cu(II)–DDC complex.Alternative samples of Cu2E2SOD or E2Zn2SOD

were prepared by titrating NaAc solution containing

CuSO4 or ZnSO4 into the solution of E2E2SOD as re-

ported elsewhere [21]. All chemical regents were of A.R.

grade and were recrystallized in twice-distilled water

before use. The ionic strengths of solutions were kept

constant by using KCl. Assayed solutions containing

about 1.0� 10�5–2.0� 10�4 mol dm�3 BESOD and ablank solution (0.1 mol dm�3 KCl and 0.05 mol dm�3

phosphate buffer (ca. pH 7.4)) were used for the elec-

trochemical experiments.

2.2. Electrochemical measurements

All electrochemical measurements were carried out

on an EG&G PARC 270 electrochemical analyticalsystem. In CV experiments, a M303 HME as the

working electrode, a saturated calomel electrode (SCE)

as the reference electrode, and a platinum coil wire as

the auxiliary electrode were used. The potentials were

measured vs the saturated calomel electrode. The inner

resistance of the cell was complemented by the appara-

tus. Each measurement was repeated several times and

the redox potentials were found to be reproduciblewithin �5 mV. In the CV experiments the blank solution

was added to a 10 cm3 cell, and then deaerated with a

slow stream of super-pure nitrogen for ca. 15 min at 25

�C. A weighed sample of SOD was added to the cell to

give a predetermined concentration, and deaeration was

continued for 10–15 min before applying the voltage.

The formal or redox half-wave potentials E1=2 were

obtained approximately from (Epa þ EpcÞ=2. In the NPP,the dropping mercury electrode with a drop time of 4 s

was used to replace HME. The NPP experiments were

carried out with a sampling time of 50 ms, an initial

voltage of )0.3 V and a return voltage of )1.25 V.

The adsorption of BESOD on a HME with an area of

0.035 cm2 was assayed by double potential-step chro-

nocoulometry [23,24]. The HME was held in solution

for several minutes before applying the potential step.The electrode potential was stepped from )0.3 to )0.8 V

for a time s (10 s) and then stepped back to )0.3 V to

allow the redox reaction of Cu(II) in BESOD to occur.

Q, the total charge passed was measured as a function of

time. Plotting Qf (the charge passed in the positive di-

rection at t5s) vs. t1=2, and Qr (in the opposite direction

t > s) vs. H1=2ðH ¼ s1=2 þ ðt � sÞ1=2 � t1=2Þ on the same

graph, two straight lines with intercepts Q0f ;Q

0r and

slopes Sf and Sr were obtained, respectively. The amount

of adsorbed BESOD (C) could be determined from

nFAC ¼ ðQ0f � Q0

r Þ=ð1� a0Þ; ð1Þwhere C is the amount of adsorbed BESOD (mol cm�2),

A is the area of the HME (0.035 cm2), a0 is a constant(0.069), n ¼ 1 for the redox reaction of copper(II) in

BESOD, F ¼ 96500 C. The diffusion coefficient D (cm2

s�1) was calculated from the slope Sf as follows

Sf ¼ 2nFcD1=2s1=2p�1=2: ð2Þ

W. Qian et al. / Journal of Electroanalytical Chemistry 568 (2004) 143–149 145

The slope ratio Sr=Sf can be calculated from:

Sr=Sf ¼ 1þ a1nFAC=Qc; ð3Þwhere Qc is the total charge arising from the diffusion of

BESOD during the forward step; the value of a1 is

usually taken as 0.97.

In the determination of pKa, the PESOD was pre-

pared by the previous method [11]. In order to ensureestablishment of slow equilibrium between SOD and

protons in the solution, each assayed solution was dia-

lyzed against a buffer solution with a predeterminate pH

value for several hours. The relationship between E1=2

and pKa of reduced SOD was determined by the equa-

tion

� logð10DE=0:059 � 1Þ ¼ pHþ pKa; ð4Þwhere DE ¼ E1=2 � Elim, Elim denotes the pH-indepen-

dent half-wave potential attained at high pH values.

3. Results and discussion

3.1. Cyclic voltammetric behavior

The cyclic voltammogram of a solution of 1.2� 10�4

mol dm�3 BESOD at pH 7.4 on a hanging mercury

electrode is shown in Fig. 1. It shows two pairs of well-

defined cathodic and anodic peaks with peak potentials

E1pc ¼ �0:680 V, E1

pa ¼ �0:610 V and E2pc ¼ �1:140 V,

E2pa ¼ �0:920 V. The formal redox potentials of the two

pairs of peaks were E11=2 ¼ �0:645 V and E2

1=2 ¼ �1:030V, respectively. The peak potential separations of the

cathodic and anodic peaks were DE1 ¼ 70 mV,DE2 ¼ 220 mV. The electrochemical process was irre-

versible. The plot of Ipc or Ipa against the scan rate mshowed a straight line, which denoted that the electrode

process possessed adsorptive characteristics under CV

Fig. 1. Cyclic voltammogram of BESOD on HME, scan rate 0.10 V

s�1, concentration of BESOD is 1.2� 10�4 mol dm�3 in 0.10 mol dm�3

KCl solution and 0.05 mol dm�3 phosphate buffer at pH 7.4. 2. blank

solution.

conditions. The magnitudes of the two pairs of peaks

did not change during repeated potential scanning after

standing the HME in 1.2� 10�4 mol dm�3 BESOD

solution for a few seconds. The electrode with adsorbed

BESOD was then removed from the BESOD solution,washed with water, and transferred into pure supporting

electrolyte solution to record the CV curves; the peaks

persisted for several minutes without significant decre-

ment. This means that adsorption of BESOD on the

electrode surface is irreversible. In Fig. 1 the separation

of each pair of peaks is not equal to zero, implying that

the surface reaction is a slow reaction. The adsorptive

behavior of BESOD on HME is similar to that of PE-SOD [18].

The dependence of the peak currents on the concen-

tration of BESOD was investigated in the range of

concentrations from 1.0� 10�5 to 2.0� 10�4 mol dm�3.

A plot of I1pa=c and I1pc=c vs the bulk concentration of

BESOD c showed that in the initial stages, the values of

I1pc=c and I1pa=c decreased sharply with increase of the

bulk concentration of BESOD, and then tended to aconstant value at a concentration around 1.0� 10�4 mol

dm�3 for both I1pc and I1pa (Fig. 2). This means that with

an increase of the BESOD concentration, the fraction of

the peak current due to adsorbed BESOD decreased and

the fraction of the peak current due to diffusion of

BESOD increased [25]. Finally the contribution of the

diffusion current predominated over the adsorption

current when the concentration of BESOD was greaterthan 1.0� 10�4 mol dm�3.

3.2. The voltammetric properties of reconstituted

Cu2Zn2SOD

In many scholars’ studies on reduction of proteins

such as insulin and bovine serum albumin etc., [26–28]

on mercury electrodes, two reversible or quasi-reversible

ig. 2. Dependences of I1pc=c (1) and I1pa=c (2) on bulk concentration of

ESOD on HME, scan rate 0.1 V s�1.

F

B

146 W. Qian et al. / Journal of Electroanalytical Chemistry 568 (2004) 143–149

voltammetric peaks or polarographic waves were often

observed at potentials of approximately )0.6 and )1.0 V(SCE) which have been attributed to reduction of the

disulfide bonds in the hydrophobic and the hydrophilic

regions of the protein, respectively [15,29]. In themonomer of Cu2Zn2SOD there is one disulfide bond

and one unbound cystine sulfhydryl group. In order to

assign carefully two pairs of peaks in Fig. 1, the vol-

tammetric properties of E2E2SOD, Cu2E2SOD and

E2Zn2SOD were studied. The reconstituted products

have the following properties: (1) In the CV diagram of

E2E2SOD (Fig. 3(a)) no redox response was seen in the

range of )0.2 to )1.3 V, when the concentration ofE2E2SOD was changed from 1.5� 10�4 to 8.6� 10�8

mol dm�3. No peaks were seen either, implying that the

disulfide or sulfhydryl bonds in SOD have not been re-

duced [29]. Therefore, the two pairs of peaks in Fig. 1

should be assigned to reduction of Cu2þ and Zn2þ, re-spectively, in Cu2Zn2SOD on HME; (2) The CV dia-

gram of Cu2E2SOD obtained by adding Cu2þ ion to the

E2E2SOD solution was similar to that obtained by ex-tracting Cu2þ from Cu2Zn2SOD. In the CV diagrams,

only one pair of peaks was displayed (Fig. 3(b)), its E1=2

()0.635 V) shifted slightly compared with that of native

Cu2Zn2SOD and the electrode reduction of Cu(II) in

Cu2E2SOD was a reversible process with adsorption

characteristics; (3) With addition of Zn2þ to the solution

of Cu2E2SOD, the CV diagram obtained exhibited a

new pair of peaks located at ca. )1.0 V corresponding tothe redox peaks of Zn(II) in Cu2Zn2SOD; (4) The CV

Fig. 3. Cyclic voltammograms of derivatives of BESOD: (a) E2E2SOD;

(b) Cu2E2SOD; (c) E2Zn2SOD. Concentration of all derivatives is

1.0� 10�4 mol dm�3, the other conditions are the same as in Fig. 1. It

should be noted that the cathodic current is plotted positive upwards.

diagram of E2Zn2SOD solution prepared by extracting

Cu2þ ion from Cu2Zn2SOD (Fig. 3(c)) also displayed a

pair of peaks corresponding to the redox peaks of Zn(II)

in E2Zn2SOD. The CV behavior of the reconstituted

derivative was independent of the method of reconsti-tution. The CV diagram of the reconstituted solution

with re-addition of the metal ions previously removed

was similar to that of the solution of native Cu2Zn2SOD

before reconstitution. The binding between metal ions

and the apoenzyme is very interesting, and the

Cu2Zn2SOD looked like a supramolecule, which was

composed of the apoenzyme as a host and the metal ion

as a guest [30]. Therefore apart from its catalytic func-tion, Cu2Zn2SOD was suggested to play a role as a

copper-transport and/or storage protein [31–33], these

experimental results give important clues to this role.

3.3. Normal pulse polarography

We investigated the reduction of adsorbed BESOD

on a dropping mercury electrode by polarography forfurther understanding of the behavior of adsorbed BE-

SOD on the electrode. Fig. 4 shows the NPP diagram of

BESOD solution. It is a remarkable fact that two well-

defined polarographic waves were observed in both the

forward and the reverse potential scans. The formation

of two waves resulted from the adsorption and succes-

sive reduction of SOD on the dropping mercury elec-

trode as described by the cyclic voltammetry. For thetwo waves, the limiting current of the forward scan was

not equal to that of the reverse scan, thus indicating the

instability of the reduced BESOD on the mercury elec-

trode. The half-wave potentials in the forward and re-

Fig. 4. The normal pulse polargraph: (1) forward scan; (2) reverse scan.

Concentration of BESOD is 1.20� 10�4 mol dm�3 in 0.1 mol dm�3

KCl solution and 0.05 mol dm�3 phosphate buffer at pH 7.0, sampling

time 50 ms, mercury drop time 4 s. Inset: plot of E vs. log½ðId � IÞ=I�for the first wave of 1. All I values in inset were obtained by sub-

stracting the background from the measured currents at the same

potentials.

Table 1

Electrochemical data of BESOD adsorbed on a mercury electrode, 25 �C

Methods pH E11=2 (V) E2

1=2 (V) 106D=cm2 s�1

CV 7.4 )0.645 )1.030NPP 7.0 )0.603 (forward) )0.955 0.74

)0.603 (reverse) )0.935Chronocoulometry 0.75

Fig. 5. A plot of amount of adsorption of BESOD on HME vs. con-

centration, solutions contained 0.1 mol dm�3 KCl and 0.05 mol dm�3

phosphate buffer, pH 7.4.

W. Qian et al. / Journal of Electroanalytical Chemistry 568 (2004) 143–149 147

verse scans were the same for the first wave (Table 1).

This means that the electrode process is reversible. The

half-wave potential for the second wave in the forward

scan ()0.955 V) was not equal to that in the reverse scan

()0.935 V), which means that the second electrode

process has poor reversibility.

For the first wave in the forward scan, the inset shows

the plot of log½ðId � IÞ=I � vs. electrode potential E atpH¼ 7.0; this was a straight line with a slope of 0.054

and from the slope, the apparent number of electron

involved in the electrode process is calculated to be

unity. The limiting current of the second wave in the

forward scan is about twice that of the first, giving

E1=4 � E3=4 ¼ 63 mV which is greater than that for a

reversible two-electron reduction process. From these

results, the first wave could be assigned to the reversibleone electron reduction of Cu(II) in SOD and the second

wave is assigned to an irreversible two-electron reduc-

tion of Zn(II) in BESOD. The assignation of the second

wave will be discussed further in the light of the results

of double potential-step chronocoulometry. The diffu-

sion coefficient of BESOD is obtained as 7.4� 10�7 cm2

s�1 in a solution of 1.6� 10�4 mol dm�3 BESOD. This

value is of the same order as that of PESOD18

(2.25� 10�7 cm2 s�1) determined by polarography.

3.4. Double potential-step chronocoulometry

The adsorption of BESOD was estimated by double

potential-step chronocoulometry [23,24]. A typical

cluster of straight lines was obtained for various bulk

concentrations by plotting Qf vs. t1=2 and Qr vs. H (seeSection 2.2), from which the intercepts Q0

f obtained were

much higher than those of Q0r , implying that adsorption

of native BESOD was predominant over that of reduced

BESOD. Thus it is reasonable to neglect adsorption of

reduced SOD for the calculation of the amount of ad-

sorption of SOD (C) over the potential range of the

Cu(II)/Cu(I) couple using Eq. (1). The calculated slope

ratio values of Sr=Sf from Eq. (3) were in good agree-ment with that obtained by plotting Qf vs. t1=2ðSfÞ andplotting Qr vs. HðSrÞ. This confirms that our assumption

is reasonable. The dependence of the amount of ad-

sorption of SOD on its bulk concentration is shown in

Fig. 5. Full coverage of SOD molecules on the surface of

electrode was apparently reached at the concentration of

1.02� 10�4 mol dm�3 SOD. The maximum adsorption

Cs was obtained as 9.20� 10�12 mol cm�2. By plotting

C=Cs vs. c, the adsorption isothermal curve obtained

deviated greatly from the Frumkin equation, but was

basically in accordance with the Langmuir equation

h=ð1� hÞ ¼ kc, where h ¼ C=Cs, k ¼ 8:96� 104. The Cs

corresponds to monolayer adsorption of closed-packed

BESOD, from which the average area of each adsorbed

BESOD molecule was obtained as 1806 �A2. The result of

X-ray structure analysis indicated that the dimer of

BESOD is an elongated ellipsoid about 33 �A wide and

67 �A long [34]. Therefore the area of the ellipsoid is ca.

1736 �A2. This is in good agreement with our result. The

value of the diffusion coefficient for BESOD calculatedfrom Eq. (2) is 0.75� 10�6 cm2 s�1 at 1.0� 10�4 mol

dm�3 BESOD concentration. This value is in accord

with that determined by NPP (0.74� 10�6 cm2 s�1, at

1.6� 10�4 mol dm�3 BESOD).

We have assigned the first wave in the polarogram to

the reduction of Cu(II) of BESOD adsorbed on the

electrode. We also may assign the second wave in the

polarogram by double potential-step chronocoulometry.For example, from the first wave the amount of ad-

sorbed BESOD, C ¼ 8:71� 10�12 mol cm�2 in a solu-

tion of 7.38� 10�5 mol dm�3 BESOD was obtained, and

from the second wave (a potential step from )0.8 to

Fig. 6. The dependences of formal redox potential on pH for: (1)

PESOD; (2) BESOD.

148 W. Qian et al. / Journal of Electroanalytical Chemistry 568 (2004) 143–149

)1.25 V) Q0f ¼ 0:2367 lC and Q0

r ¼ 0:1119 lC were

obtained. By using Eq. (1), we can obtain the electron

number of the reduction of Zn(II) in each subunit of

BESOD as n ¼ 1:98 by using the values of Q0f and Q0

r for

the second wave. Obviously, the second wave resultedfrom the reduction of Zn(II) on the electrode.

The overall electrode process of BESOD on the

mercury electrodes was suggested as follows:

( )sol and ( )ad denote the SOD in solution and ad-

sorbed at the electrode, respectively. The reactionsproceed via electron exchange between the electrodes

and adsorbed SOD, with subsequent electron exchange

between adsorbed SOD and freely diffusing SOD. With

only the exception of the NPP wave for Cu(II) electro-

reduction, the waves are irreversible, i.e., the electrode

processes are controlled by some heterogeneous ele-

mentary step, with a possible partial control from the

diffusion step.

3.5. Protonation constant of the imidazolate bridge

In order to confirm further the feasibility of the HME

for the investigation of SOD in the absence of mediators

(or promotors), the pKa of reduced SOD was deter-

mined for comparison with that in the presence of

promotors or mediators reported by other authors[11,13]. The pH-dependence of the formal redox po-

tential E1=2 values for the Cu(II)/Cu(I) couple of PESOD

and BESOD, respectively, are shown in Fig. 6. For both

PESOD and BESOD, the E1=2 values decreased linearly

with an increase of pH from 5 to 8. The slopes of the two

straight lines were 58 and 57 mV/pH, respectively, cor-

responding to the binding of one proton to the bridging

imidazolate accompanied by the reduction of SOD. InTable 2 are shown pKa values of PESOD and BESOD

obtained by constant-current coulometric titration using

a Pt electrode [11], by cyclic voltammetry using a gold

electrode [13] and our results obtained from Eq. (4). No

matter what methods and electrodes were used, or

whether promotors or mediators were used or not,

Table 2

pKa of the bridging imidazolate of reduced BESOD and PESOD

Methods Electrodes Mediators (or prom

CV (this work) HME None

CV Au [13] 1,2-bis(4-pyridyl)et

Coulometric titration Pt-coil [11] Methyl viologen

consistent results were obtained. These facts prove that,

in the absence of promotors or mediators, the hangingmercury electrode (or dropping mercury electrode) can

be used for the study of SOD.

4. Conclusion

(1) The electrochemical behavior of BESOD on the

HME was studied. Two pairs of peaks in the CV di-agram were attributed to the redox peaks of cop-

per(II) and zinc(II), respectively, not to disulfide or

sulfhydryl bonds. The electrode processes were irre-

versible and possessed adsorptive characteristics.

(2) The adsorption of BESOD was estimated by double

potential-step chronocoulometry, and corresponded

to monolayer adsorption. The average area of each

molecule of adsorbed SOD is in good agreementwith that determined by X-ray structure analysis.

(3) The diffusion coefficient of SOD and the apparent

electron numbers of the reduction of Cu(II) and

Zn(II) were obtained by NPP and double poten-

tial-step chronocoulometry, respectively. They are

consistent with each other.

(4) The protonation constants of the imidazolate bridge

in reduced BESOD and PESOD were determined byCV on the HME. They are in agreement with those

obtained in the presence of promotors or mediators.

Acknowledgements

This project was supported by the Natural Science

Foundation of China.

oters) BESOD PESOD

8.05� 0.15 8.12� 0.2

hane 8.2� 0.6 8.12� 0.3

8.15� 0.2 8.04� 0.14

W. Qian et al. / Journal of Electroanalytical Chemistry 568 (2004) 143–149 149

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