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Voltammetric Investigation of Zinc Release from MetallothioneinsModulated by the Glutathione Redox Couple and Separated with aPorous MembraneLin Liu,a Julei Yang,a Ning Xia,a Jianxiu Wang,a* Feimeng Zhoua, b

a School of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, 410083, P. R. Chinab Department of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, CA 90032, USA*e-mail: wangjianxiu1969@yahoo.com

Received: May 5, 2008Accepted: July 17, 2008

AbstractGlutathione (GSH), in addition to serving as a redox buffer in cellular environment, has been suggested as amodulator in metal regulation and homeostasis by metallothioneins (MTs). The interactions of MTs with both GSHand its oxidized form GSSG have been shown to govern the direction of metal transfer. Common methods for thedetermination of zinc release from MTs modulated by GSH/GSSG either involve radioactive species or enzymes orare labor-intensive. In this study, upon separation of Zn2þ from the reaction mixture of MTs and GSH with acentrifugal filter membrane, differential pulse voltammetry (DPV) was used for the Zn2þ quantification. The sameapproach is extended to the studies of metal transfer between Zn7MT with a GSH/GSSG mixture and that betweenZn7MT with GSSG. The concomitant conversion between the free thiol and disulfide bonds was confirmed with UV-vis spectrophotometry. The results demonstrate that GSSG, GSH, and the GSH/GSSG mixture all modulate zincrelease from Zn7MT. The percentage of zinc release increases in the order of GSH, GSSG, and the GSH/GSSGmixture. The new approach is demonstrated to be well suited for investigation of redox regulation of MT and itsreaction with zinc-containing enzymes.

Keywords: Metallothionein, Glutathione redox couple, Zinc release, Voltammetry, Spectrophotometric assays

DOI: 10.1002/elan.200804309

1. Introduction

Metallothioneins (MTs) are a class of low molecular weight(6 – 7 kDa), cysteine-rich (up to 30% of the total aminoacids) metalloproteins [1]. The tertiary structure of MTsadopts a dumbbell-like shape comprising two clusters inwhich the metal ions are coordinated by bridging andterminal cysteine thiol ligands [2, 3]. It is generally believedthat MT exerts its functions in regulating essential metals,detoxifying heavy metals, scavenging free radicals, andcontrolling the intracellular redox potential [4, 5].

Glutathione, a tripeptide with a single cysteine residue, isan important redox species in cytoplasm that plays a centralrole in protecting cells against high levels of heavy metalions and regulating various cell functions [6 – 14]. Thestudies on the interaction between MTs and the reducedform (GSH) or oxidized form (GSSG) of glutathione havebeen carried out extensively [15 – 21]. GSH has been foundto form a complex with both vertebrate and invertebratecopper-metallothioneins (Cu-MTs), which can release cop-per in the form of Cu-GSH. Similarly, GSH interacts withCdZnMT-I from blue crab hepatopancreas and CdZnMT-IIfrom rabbit liver. Formation of these complexes may resultin zinc or cadmium release in a similar way to the above-mentioned copper release [15, 16]. Maret et al., by measur-

ing the concomitant radioactive 65Zn release from MTs [19,20], showed that GSSG could release 20% of the total zincfrom MT via a zinc-thiol/disulfide interchange mechanism.The kinetic studies of the reconstitution of apo-sorbitoldehydrogenase (SDH) with zinc released from MT byVallee and co-workers indicate that the GSH/GSSG redoxcouple modulates zinc transfer from MT to a zinc acceptor[18]. GSH plays a dual role: in the absence of GSSG, itinhibits the zinc release from MT, whereas in the presence ofGSSG, it stimulates the reaction between MTand GSSG andincreases the number of zinc ions transferred. The reactionbetween MT and GSSG was monitored by spectrophoto-metric assays of zinc released from MT with zinc-complex-ing dyes such as 4-(2-pyridylazo) resorcinol (PAR) and 2-carboxy-2’-hydroxy-5’-sulfoformazylbenzene (zincon) [21].However, these methods either involve radioactive speciesor enzymes or are labor-intensive. As a result, a quick andstraightforward method is desired for the quantification ofzinc released from MTs in the presence of the glutathioneredox couple.

Voltammetric techniques are simple, sensitive, and inex-pensive to implement [22]. Typical examples for the studiesof MTs include the use of cyclic voltammetry, square wavevoltammetry and differential pulse voltammetry to examinethe redox properties of MTs and the redox-induced metal

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release from MTs [23 – 30]. For example, elucidation of theMT metal transfer process under various redox conditionsand quantification of the metal release associated with theMT redox reactions at thin mercury film electrodes havebeen reported [23, 30]. Several other groups also useddropping mercury electrodes to examine the redox proper-ties of MTs and their isoforms [26, 27, 29]. We have utilizedNafion-coated mercury film electrodes to explore the redoxproperties of rabbit liver MTs containing both Zn and Cdions and Zn7MT [24]. Due to the absence of the intermetalliccompound formed between Cd and Zn, the amount of themetals released from MTs can be quantified. However, tothe best of our knowledge, no attempt has been made tovoltammetrically quantify zinc release from MTs modulatedby the glutathione redox couple. In the present study, weused thin mercury film electrodes to determine amounts ofzinc released upon respectively reacting MTs with GSH,GSSG, and the GSH/GSSG mixture. Upon separation ofZn2þ from the reaction mixtures with a centrifugal filtermembrane, Zn2þ eluted through the membrane weredeposited into the electrode and quantified accurately.The accuracy was further confirmed with atomic absorptionspectrophotometry (AAS) and the concomitant change ofthe sulfhydryl groups in MTs was characterized with UV-visspectrophotometry and voltammetry.

2. Experimental

2.1. Reagents and Instruments

Reduced and oxidized forms of glutathione (GSH andGSSG, respectively), tris(hydroxymethyl)aminomethanehydrochloride (Tris-HCl), 5,5’-dithiobis(2-nitrobenzoicacid) (DTNB), and tris(2-carboxyethyl)phosphine (TCEP)were obtained from Sigma (St. Louis, MO). Rabbit liver Zn7

MT was acquired from Human Lugu Biotech Co., Ltd(Changsha, China). Mercury and zinc standard solutionswere purchased from Aldrich Chemical Company, Inc.(Milwaukee, WI). Microcon YM-3 centrifugal filter unitswere purchased from Millipore Corp. (Belleria, MA). Otherreagents are all from commercial sources with analyticalpurity or better and used as received. DTNB was dissolvedin 20 mM Tris-HCl buffer (pH 8.0). Other solutions wereprepared with a 20 mM Tris-HCl buffer (pH 7.4).

Quantification of zinc release was carried out using a CHI660B electrochemical workstation (Austin, TX) and aWXY-402C flame atomic absorption spectrophotometer(Shenyang Analytical Instrument Co., LTD, China). Forthiol determination, a UV-vis spectrophotometer (Shi-madzu Co., Japan) and the CHI 660B electrochemicalworkstation were used.

2.2. Electrodes

The working electrode was a glassy carbon disk (GC) with adiameter of 3 mm. A platinum wire and a Ag/AgCl

electrode were used as the auxiliary and the referenceelectrodes, respectively. Prior to each measurement, the GCelectrode was polished with alumina slurry down to 0.05 mmon a polishing cloth, followed by sonication in water andethanol. Mercury films were deposited onto the GCelectrode surface by holding the electrode potential at�0.4 V for 300 s in a N2-degassed 1% HNO3 solutioncontaining 5 mM Hg2þ. All measurements were conductedat the ambient temperature (25� 1 8C).

2.3 Procedures

2.3.1. Quantification of Zinc Release from Zn7MT

Rabbit liver Zn7MT was incubated with GSH, GSSG or aGSH/GSSG mixture in a 20 mM Tris-HCl buffer (pH 7.4)for 1 h, respectively. The molar ratio of MT: GSH: GSSGused is 1: 100: 200. The optimized ratio of GSH/GSSG waschosen according to the literature [18]. The reactionmixtures were filtrated with a 12.3-mm-diameter YM-3membrane at 13 000 rpm for 20 min. Zn2þ ions elutedthrough the membrane were determined by differentialpulse voltammetry (DPV) and atomic absorption spectro-photometry (AAS). In the case of the DPV detection, apredetermined deposition time at �1.3 V was used todeposit zinc at the mercury film prior to the potential scanbetween �1.3 and �0.8 V. Amounts of zinc released fromMT were determined based on the standard additionmethod.

2.3.2. Characterization of Sulfhydryl Groups in MTs

Before each assay, all the MT samples were washed fourtimes with Tris-HCl buffer (pH 7.4). Spectrophotometricthiol assays were conducted with the Ellman method byexamining the absorbance change of the intensely coloredproduct at 412 nm [31, 32]. The concomitant change ofsulfhydryl groups in the MT samples was also characterizedby voltammetry.

3. Results and Discussion

3.1. Voltammetric Quantification of Zinc Release fromZn7MT

The unique cluster structure of MT allows it to bind zinctightly with both bridging and terminal thiolate ligands [2,3]. However, the coordinated zinc ions may become mobilein the presence of various oxidizers or at low pH [18, 20, 21,33]. Regulation of zinc by MT that is also modulated by theglutathione redox couple is investigated in our work.Treatment of Zn7MT with GSH or GSSG or the GSH/GSSG mixture for 1 h caused zinc to release from Zn7MT.The above mixtures were filtered with an YM-3 membranewith a pore size of about 1 nm which has a molecular weightcutoff of 3 kDa. The molecular weight of Zn7MT is 6 –

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7 kDa, while those of zinc, GSH, and GSSG are all below3 kDa. Therefore, MT remained on top of the filtermembrane whereas GSH, GSSG, and released metal ionswill elute through the pores of the membrane and becomeseparated from the MT species. Amounts of zinc releasedwere determined by DPV, and spectrophotometric thiolassays of the concomitant change of sulfhydryl groups in theMT samples were carried out.

Figure 1 shows DPVs of the anodic stripping of Zn2þ

released from Zn7MT, Zn7MT/GSH, Zn7MT/GSSG andZn7MT/GSH/GSSG solutions and eluted through themembranes. The anodic peaks at about �1.125 V areattributable to the stripping of Zn deposited into the Hgfilm [23, 24]. As can be seen, only a small anodic peak wasobserved in the case of Zn7MT alone (curve 2), suggestingthat in the absence of a redox species, the metal ions arelargely withheld by MT. The small amount of zinc released incurve 2 can be ascribed to the breakage of the thiolate bondsin the MT clusters brought about by the continuous�interchange� between the bridging and terminal sulfhydrylgroups [19]. In the presence of GSH, an increase of theamount of zinc released from Zn7MT was observed (curve3), with the peak current being 2.7 times greater than that incurve 2. With the introduction of GSSG (curve 4) or theGSH/GSSG mixture (curve 5) to the Zn7MT solution, asubstantial increase of the anodic peak currents (6.2 and 6.4times greater than that in curve 2, respectively) wasobtained. We note that the slight change of the anodicpeak potential is due to the formation of Zn-GSH or Zn-GSSG complex [34]. Zn-GSH complex formation may beascribed to the coordination of zinc with the sulfur atom andthe amino group of GSH. While in the case of Zn-GSSGcomplex, the coordination is probably through the aminoand a-carboxylate groups of GSSG. To examine theinfluence of Zn-GSH or Zn-GSSG complex formation onthe voltammetric responses, we performed an experiment inwhich the DPVs of several zinc standards were collected inthe absence and presence of GSH, or GSSG, or the GSH/GSSG mixture (data not shown). The stripping signals of thezinc standards decreased by 4.5%, 2.5%, and 8.6% in thepresence of GSH, GSSG, and the GSH/GSSG mixture,respectively. Since DPV measures the free Zn2þ ions insolution, the above results indicate that zinc ions elutedthrough the membrane are not strongly complexed by GSHor GSSG or zinc ligated by GSH or GSSG can also bereduced and accumulated into the Hg film electrode.

Previous studies have suggested that the GSH/GSSGredox couple could result in the zinc release from MTs [18 –21]. However, due to the difference in reaction conditions ordetection methods, the numbers of zinc ions released haveremained inconsistent. Using a radiochromatographic tech-nique, Maret determined the percentage of zinc releasedfrom MTs in the presence of GSSG to be 20% after 1 h ofreaction and nearly 100% after 6 h [19]. Vallee and co-workers investigated the reconstitution of zinc depletedSDH with MTs [18] and their results indicated that one ofthe seven zinc atoms in MTs can be transferred in theabsence of GSH and GSSG. However, the amount of zinc

atoms transferred increases to four when the GSH/GSSGredox couple is present. GSH alone slightly inhibits the zinctransfer, but strongly facilitates the reaction between MTand GSSG. In order to voltammetrically determine themetal release, solutions containing Zn7MT in the presenceof GSH, or GSSG or the GSH/GSSG mixture after reactionfor 1 h were filtrated. Based on the standard additionmethod (Fig. 2), we found that the concentrations of zincreleased upon respectively reacting MTs with GSH, GSSG,and the GSH/GSSG mixture were determined voltammetri-cally to be 23.0 mM, 134.4 mM, and 139.7 mM, respectively.The percentage of zinc eluted through the membraneincreases in the order of GSH (curve a, 16.5%), GSSG(curve b, 96.0%), and the GSH/GSSG mixture (curve c,99.7%). We also conducted a series of experiments in whichrabbit liver Zn7MT was incubated with GSH, GSSG or theGSH/GSSG mixture in a 20 mM Tris-HCl buffer (pH 7.4)for 5 min, 20 min, 40 min, 1 h, and 3 h, respectively. Theresults indicate that the percentage of zinc eluted throughthe membrane in the three cases increases with theincubation time and plateaus after 1 h. So 1 h was chosenas the most suitable time of glutathione and MT interaction.

As described previously, modulation of GSH towards zincrelease from MTs may originate from the formation of asmall amount of GSSG oxidized from GSH by O2 in solution[18]. To ensure that GSH remains in the reduced form, areductive nucleophilic reagent, such as TCEP or dithio-threitol (DTT) [35], can be added to the GSH solution. Anappreciable zinc anodic peak current was observed with theaddition of TCEP to the mixed solution of MTs and GSH(curve 3, Fig. 3). In comparison with the MT alone solution(curve 1), the higher stripping signal in the presence ofTCEP in curve 2 might be resulted from the weak binding

Fig. 1. Differential pulse voltammograms (DPVs) at thin mer-cury film electrodes of 20 mM Tris-HCl buffer (curve 1) andeluents through the membrane from the Zn7MT (curve 2), Zn7

MT/GSH (curve 3), Zn7MT/GSSG (curve 4), and Zn7MT/GSH/GSSG (curve 5) solutions. The concentrations of MT, GSH, andGSSG were 20 mM, 2 mM, and 4 mM, respectively. Before eachassay, the eluents were diluted 20-fold with Tris-HCl buffer(pH 7.4). A deposition time of 200 s at �1.3 V was used. Pulseheight: 50 mV; pulse width: 50 ms. The arrow indicates the scandirection.

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between TCEP and Zn2þ [36], which could have slightlyhindered the reconstitution of MTs with free zinc ions in thefiltration process. Thus, it is reasonable to conclude thatGSH could alone facilitate zinc release from MTs. Theabovementioned zinc release process modulated by GSH issimilar to that involved in the copper release [15, 16] butdifferent from that reported by Vallee et al. [18]. Thisdiscrepancy can be rationalized on the basis that in Vallee�swork [18] binding of GSH to apo-SDH may have affectedthe activity of apo-SDH and the follow-up reconstitution ofapo-SDH with free zinc ions from MT-II. Based on theabove results and those reported previously [15, 16, 37], wepropose that zinc release from MTs modulated by GSH isdue to the competition of cysteine residues in GSH for zincions in MTs. Such a competition process may also increasethe turnover rate of MTs and lead to a conformationalchange of the MT molecule [16], providing reactive thiols inMTs (especially those exposed to the solvent) for furtherreaction with GSSG [18].

We conducted atomic absorption spectrophotometric(AAS) measurements to further verify the amounts ofZn2þ determined by DPVs separated from the varioussolutions or the accuracy of the percentages of the zincrelease. The concentrations of zinc released from the Zn7

MT/GSH, Zn7MT/GSSG, and Zn7MT/GSH/GSSG solu-tions were determined to be 33.3 mM, 124.0 mM, and128.2 mM, respectively. The percentages of zinc releases inthe three cases were found to be 23.8, 88.6, and 91.6%,respectively. These values are in good agreements with thosemeasured by DPV, suggesting that the DPV measurementsare reliable. However, for the two methods involved, DPV ismore sensitive than AAS [38].

3.2. Characterization of Sulfhydryl Groups in MTs

The extent of Zn2þ released from MTs that is modulated bythe glutathione redox couple is accompanied by a change ofthe redox states of the sulfhydryl groups in MTs, as alluded

above and suggested in literature [19]. We thereforeattempted to determine the concomitant conversion be-tween the free thiol and disulfide bonds using an UV-visspectrophotometric assay. It is commonly known thatDTNB (Ellman�s reagent) is a strong electrophilic reagentwhich can react with the metal-thiolates in metalloproteinsincluding MTs [32]. Upon respective reactions with GSSGand the GSH/GSSG mixture, the MT-containing solutionsabove the filter membrane were collected and assayed withDTNB (Fig. 4). In comparison with curve 3, it is obvious thatMT-SG, the putative mixed disulfide with glutathione [19],was formed (curve 2), which results in a decrease ofsulfhydryl groups in MT. Although binding of MTs toGSSG produces GSH simultaneously, due to its lowermolecular weight, GSH has permeated the filter membraneand was not in the MT-containing solution. The almost

Fig. 2. Plots of the anodic peak currents (ipa) versus concentrations of Zn2þ eluted through the membrane from Zn7MT/GSH (a), Zn7

MT/GSSG (b), and Zn7MT/GSH/GSSG (c) that are spiked with different amounts of Zn2þ: 0, 3, 6, 9, 12, and 15 mM. The concentrationsof MT, GSH and GSSG were 20 mM, 2 mM and 4 mM, respectively. Before each assay, the eluent in (a) was diluted 40-fold with 20 mMTris-HCl buffer (pH 7.4), while in (b) and (c), the eluents were diluted 80-fold. Deposition times of 80 s in (a) and 150 s in (b) and (c) at�1.3 V were used. The linear regression equations are ipa (mA)¼ 0.57þ 0.99 CZn2þ (mM) for plot (a) (R2¼ 0.9982), ipa (mA)¼ 2.47þ 1.47CZn2þ (mM) for plot (b) (R2¼ 0.9998), and ipa (mA)¼ 2.48þ 1.42 CZn2þ (mM) for plot (c) (R2¼ 0.9995).

Fig. 3. DPVs of the eluents from Zn7MT (curve 1), Zn7MT/TCEP (curve 2) and Zn7MT/GSH/TCEP (curve 3). The concen-tration of Zn7MT was 20 mM. The molar ratio of Zn7MT/GSH/TCEP was kept at 1 :100 :10. Before each assay, the eluents werediluted 20-fold with 20 mM Tris-HCl buffer (pH 7.4). A deposi-tion time of 200 s at �1.3 V was used. The other experimentalconditions are the same as those in Figure 1.

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indiscernible absorption in curve 1 indicates that GSHprimes MT for the reaction with GSSG through theformation of MT-SG and the released metal ions. MT-SGformation thus results in breakage of the original complexformed between GSH and cysteine clusters of MT [15, 16].We should note that the decrease of the sulfhydryl groups inthe MT samples from Zn7MT/GSSG (curve 2), and Zn7MT/GSH/GSSG solutions (curve 1) is accompanied by anincrease of the amount of Zn2þ released. As an alternative,the concomitant change of sulfhydryl groups in the MTsamples was monitored by examining the electrochemicalsignal of thiol groups present in the MT-containing solutions

at thin mercury film electrodes. The signal at �0.62 Vrelates to the interaction of thiol groups of MTs withmercury (Fig. 5) [39 – 42]. A similar trend in the concomitantconversion between the free thiol and disulfide bonds wasobtained by UV-vis spectrophotometric assay and electro-chemical characterization. The above results providedadditional evidence for the occurrence of the zinc releasefrom MTs that is modulated by the glutathione redoxcouple.

4. Conclusions

In this study, voltammetry has been used to quantify zincrelease from Zn7MT modulated by the glutathione redoxcouple. By choosing the appropriate filter membrane, weshow that metal ions released can be straightforwardly andquantitatively separated from MTs. Zinc ions released fromZn7MT via filtration can be analyzed with DPV andquantified based on the standard addition method. Thecontent of Zn2þ released and the concomitant change ofsulfhydryl groups in MTs were also determined with AASand UV-vis spectrophotometry, respectively. A similar trendin the amount of zinc released from MTs was obtained byDPV and AAS, with the percentage of zinc releasedincreases in the order of GSH, GSSG, and the GSH/GSSGmixture. The much greater percentage values of zincreleased than those reported previously [18 – 21] may beascribed to the treatment of the reaction mixtures via theeffective and simple filtration and the reliability of thefollow-up determination. The method described hereinobviates the use of radionuclides or expensive enzymes anddoes not require chromatographic separations. The inter-action of Zn7MT with glutathione redox couple has physio-logical implications regarding the role of glutathione in zincmetabolism and bioavailability.

5. Acknowledgements

Partial support of this work by the National Natural ScienceFoundation of China (No. 20775093) is gratefully acknowl-edged.

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