bioelectrochemical dehalogenations via direct electrochemistry of poly(ethylene oxide)-modified...

1388-2481/99/$ - see front matter q1999 Elsevier Science S.A. All rights reserved.PII S1388- 2481 (99 )00125 -3

Thursday Nov 11 01:03 PM StyleTag -- Journal: ELECOM (Electrochemistry Communications) Article: 135

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Electrochemistry Communications 1 (1999) 609–613

Bioelectrochemical dehalogenations via direct electrochemistry ofpoly(ethylene oxide)-modified myoglobin

Martin Wright, Michael J. Honeychurch, H. Allen O. Hill *Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR, UK

Received 17 August 1999; received in revised form 1 October 1999; accepted 4 October 1999

Abstract

Catalytic reductive dehalogenation of hexachloroethane (HCE) was shown to be possible using a graphite electrode coated with a film ofthe poly(ester sulfonic acid) containing myoglobin (Mb). The effectiveness of the dehalogenation was limited by the solubility of HCE inaqueous solutions. In order to produce an electrode that could dehalogenate HCE in non-aqueous solutions, Mb was chemically modified byaddition of poly(ethylene oxide) (PEO). The PEO–Mb–AQ29D modified electrode was found to be suitable for the reductive dehalogenationof HCE in ethanolic solutions. The catalytic response was shown to be linearly dependent on the bulk concentration of HCE. q1999Elsevier Science S.A. All rights reserved.

Keywords: Myoglobin; Poly(ethylene oxide); Dehalogenation; Hexachloroethane; Protein

1. Introduction

Halogenated hydrocarbons are extensively used as sol-vents and in synthesis and as such make up the largest groupon the EPA list of priority pollutants [1]. Some of thesecompounds are acutely or chronically toxic, being suspectedcarcinogens and teratogens [2], and many persist in the envi-ronment. This makes it very desirable to be able to quicklyand efficiently detect these compounds. The principal meth-ods of detecting these compounds presently are by HPLC andGC, both of which are time-consuming and costly.

The aim of this work is to study the reductive dehalogen-ation of simple organic molecules by the heme-containingprotein myoglobin (Mb), with the possible future use of thistechnology in the detection of halogenated hydrocarbons inthe environment.

Because many halogenated hydrocarbons are immiscibleor insoluble in water it is necessary to make the protein stablein a non-aqueous environment. To achieve this we have mod-ified the protein surface with polyethylene oxide (PEO).PEO modification has been shown to increase the stability ofproteins in a lypophilic environment, and also to increase thethermal stability of proteins [3], which is of benefit for com-mercial biosensors.

* Corresponding author. Tel.: q44-1865-275-900; fax: q44-1865-272-690; e-mail: [email protected]

2. Experimental methods

The electrochemical experiments were carried out with anEco Chemie PGSTAT 10 Autolab. The electrochemical cellused was a standard two-compartment glass cell, with a Lug-gin capillary connecting the reference compartment to theworking compartment. The working compartment of the cellalso housed a platinum gauze counter electrode with anapproximate area of 1 cm2 that was secured to the cell wall.Aqueous electrochemistry was performed in a 2-[N-mor-pholino]ethanesulfonic acid (MES) buffer dissolved inultra-pure water (Milli-Q, Millipore) with an AgNAgClNKCl(aq) electrode as the reference electrode.

Electrochemistry in ethanol was performed with 50 mMtetrabutylammonium perchlorate as a supporting electrolyteand 2 mM MES buffer. The reference electrode was anAgNAgCl electrode in contact with a saturated solution ofLiCl in ethanol. All potentials reported herein are with respectto the NHE.

Before use each electrode was polished successively with0.3 and 0.015 mm grain size alumina/water slurry to obtaina mirror finish. The electrode was then sonicated to removeadhered alumina.

Experiments were performed in a glovebox under anaer-obic conditions (oxygen level below 2 ppm) at a temperatureof 308C.

Horse heart Mb was obtained from Sigma. To purify thenative Mb for electrochemistry it was first re-hydrated inMES

M. Wright et al. / Electrochemistry Communications 1 (1999) 609–613610


Fig. 2. UV–Vis spectra of oxidized Mb and oxidized PEO–Mb.

Fig. 1. UV–Vis spectra of reduced Mb and reduced PEO–Mb.

buffer at pH 6.0 and then passed through a 10 kDa sizeexclusion column (PD-10, Pharmacia) to remove any freeheme, eluting with pH 6.0 buffer. For polymer modificationthe Mb was re-hydrated in borate buffer at pH 9.0.

Activated PEO (a-PEO, MW 5000 kDa) was prepared bythe procedure reported by Ohno and Tsukada [3]. The a-PEO and Mb (ratio 5:1) were dissolved in 20 ml of 50 mMborate buffer at pH 9.0 containing 100 mM KCl. The mixturewas stirred at room temperature for 30 min. The reaction wasthen quenched by the addition of a 30-fold excess L-lysine.The excess a-PEO was removed from the reaction solutionby exhaustive centrifugation in a centricon tube with a 15kDa cut-off filter. During the successive centrifugation thebuffer was exchanged for pH 6.0 MES buffer (electrochem-istry) or pH 7.4 phosphate buffer (UV–Vis spectrophotom-etry). The number of a-PEO chains that had attached to theMb were estimated by SDS-PAGE (BioRad mini protean IISDS-PAGE kit). The effect of the synthesis procedure on theMb was monitored by UV–Vis spectrophotometry (Perkin-Elmer Lambda 20). Reduced deoxyMb (both PEO modifiedand native) was prepared by addition of a few grains ofsodium dithionite to the Mb solution in the glovebox.Reduced oxyMb was prepared by gently bubbling air throughdeoxyMb(FeII) solution.

Poly(ester sulfonic acid) ionomer (Eastman AQ29D),was used as a 1% w/v dispersion in water. 10 ml of a 1:1mixture of Mb solution and AQ29D dispersion was placedon the electrode surface and allowed to dry in a chilled cabinetat 48C.

3. Results and discussion

3.1. Preparation of PEO-modified myoglobin

The UV–Vis spectra of Mb are characteristic of the oxi-dation state of the heme iron and the ligands bound to theheme iron. In deoxygenated phosphate buffer the heme ironin Mb(FeIII) is a six coordinate high-spin complex with awater molecule bound at the sixth coordination site. Theaddition of dithionite reduces this complex to give Mb(FeII),which is five coordinate high-spin. In the presence of air thisvery slowly auto-oxidizes to give the ferric form, therebyallowing the spectra of oxygen-bound reduced Mb (reducedoxyMb) to be measured. The ferrous state is capable of bind-ing molecular oxygen at the sixth coordination site to give asix coordinate low-spin ferrous-oxy species. This propertyis used in muscle tissue to store molecular oxygen. TheMb(FeIII), deoxyMb(FeII), and oxyMb(FeII) all possesscharacteristic spectra. To check that the protein had not dena-tured during the PEO modification procedure UV–Vis spectrawere recorded of the oxy and deoxy reduced PEO–Mb andoxidized PEO–Mb and compared to native Mb. Figs. 1 and2 show that there was good agreement between PEO–Mb andMb spectra (allowing for concentration differences), indi-cating that there had been no denaturing of the protein in the

modification process. The UV–Vis spectrum of PEO–Mbrecorded in ethanol was the same as that recorded in aqueousbuffer indicating that PEO–Mb was stable in ethanol.

The degree of modification of the protein was determinedby SDS-PAGE as shown in Fig. 3. There are 19 lysine resi-dues in Mb and all of these are found on the external surfacewith a cluster of residues close to the heme binding pocket.This has potential implications on the electrochemistry of theprotein. If the PEO chains bind to the lysine residues sur-rounding the binding pocket it is possible that they will actas a barrier to any molecule trying to enter the pocket. Con-versely, they may help retain the integrity of the protein bypreventing the heme, which is not covalently bound to theprotein, from leaving its binding pocket. Also, the presenceof PEO chains may inhibit electron transfer by increasing thedistance between the Mb heme group and the electrode sur-face. Fig. 3 shows that the PEO-modified Mb contained dis-crete bands of approximately 25, 40, 60, and 95 kDa. Thiscan be interpreted as each band being due to 2, 5, 9, and 16PEO chains, respectively, becoming covalently bound to theMb. The density of the bands in the SDS-PAGE gives anapproximate indication of the relative distribution of each ofthe molecular weights.

M. Wright et al. / Electrochemistry Communications 1 (1999) 609–613 611


Fig. 3. SDS-PAGE of PEO–Mb: (A) standard molecular weight ladder; (B)PEO–Mb following concentration by centrifugation.

Fig. 4. CV of Mb–AQ29D in MES buffer at pH 6.0, 100 mV sy1.

Fig. 5. CV of Mb–AQ29D in MES at pH 6.0, 50 mV sy1. A catalyticreduction peak appears following the addition of 10 ml of 0.1 M HCE to thecell and increases following the second 10 ml addition.

3.2. Electrochemistry

Native horse-heart Mb displays very sluggish kinetics at abare BPG or EPG electrode [4]; however, the response canbe greatly enhanced by embedding the protein molecules inAQ29D films [5]. A typical response of a Mb–AQ29D filmis shown in Fig. 4. The CVs of Mb-AQ29D films, correspond-ing to the reduction/oxidation of the protein heme, typicallyhad symmetric peak shapes and nearly equal peak heights.

The potentials of the peaks were y165"5 mV at 100 mVsy1. This is more negative than the value of y121 mV versusNHE reported by Hu and Rusling [5] in the similar ionomerEastman AQ38D.

A plot of peak current, ip, versus sweep rate was linear,indicating either that the electroactive species was adsorbedonto the electrode surface, or that thin-layer electrochemistrywas being observed. In this context thin-layer electrochem-istry would be diffusion of protein within the thin layer ofpolymer coating the electrode.

Following characterization of the Mb–AQ29D system, 10ml aliquots of 0.1 M hexachloroethane (HCE) in ethanolwere added to the cell. Fig. 5 shows that the addition of HCEproduced a catalytic response, without the positive shift inpotential that is observed for dioxygen reduction. Additionof the solvent ethanol alone failed to produce any catalyticresponse, indicating that the response was not due to anyresidual dioxygen in the ethanolic stock HCE solution.Increasing the concentration of HCE in the cell beyond ;1mM caused the solution to become cloudy due to the solu-bility of HCE in water being reached. No further increase inthe catalytic current was observed. The reaction is assumed

to follow the mechanism described by Logan et al. [6] inwhich the initial step is the departure of the chloride ion toform the pentachloroethane carbocation which then receivesan electron from the Mb(FeII). The Mb(FeIII) is rapidlyreduced by the electrode to Mb(FeII) and the pentachloro-ethane radical receives a second electron as another chloridegroup leaves, thus forming tetrachloroethylene.

yyCl ∞Cl C–CCl ™ Cl C–CCl (1)3 3 2 3

yqe∞ x

Cl C–CCl ™ Cl C–CCl2 3 2 3

y yyCl , qex

Cl C–CCl ™ Cl CsCCl2 3 2 2

As no further progress could be made in aqueous solutionsdue to the limited solubility of HCE, ethanol was used as thesolvent. HCE, along with many other halogenated hydrocar-bons, is more miscible/soluble in ethanol than in water, so itwas hoped that the solubility problem would be overcome byusing ethanol as a solvent. However, the dehydrating effectof ethanol on Mb led to denaturing of the protein and thereforePEO–Mb was used because it has been shown [3] that the

M. Wright et al. / Electrochemistry Communications 1 (1999) 609–613612


Fig. 9. Plot of Di vs. HCE added for PEO–Mb–AQ29D in ethanol showingthe increase in current at y300 mV with each addition.

Fig. 8. CV of PEO–Mb–AQ29D in ethanol at pH 6.0, 50 mV sy1 andfollowing 10 ml additions of 0.1 M HCE in ethanol to the cell. The catalyticreduction current increases with increasing concentration of the HCE in thecell solution.

Fig. 7. CV of PEO–Mb–AQ29D in ethanol at pH 6.0, 200 mV sy1.

Fig. 6. CV of PEO-Mb–AQ29D in MES buffer, pH 6.0, 500 mV sy1.

addition of PEO chains to the surface of proteins, in particularMb, increased their stability in non-aqueous environments. Itis thought that the polymer chains surrounding the proteintrap water molecules close to the protein surface, thus helpingit to retain its hydration shell.

Initial experiments were performed in aqueous buffer toallow comparison with native Mb. A typical cyclic voltam-mogram (CV) of PEO–Mb–AQ29D in aqueous buffer isshown in Fig. 6. Typically, the peak potentials of these scanswere more positive than native Mb in AQ29D, suggestingthat the response observed for native Mb may be due to freeheme which is typically seen at slightly more negative poten-tials [4] whereas the response in this experiment was due tothe Mb-bound heme.

A plot of ip versus sweep rate was linear. It is more probablethat this indicates that the electroactive species was adsorbedonto the electrode surface rather than it being due to thin-layer behaviour. This is because the larger size of the PEO–Mb relative to native Mb would make it more likely to adsorbonto the electrode surface. Also its greater size will slow andmay even prevent diffusion through the AQ29D film.

The electrochemistry of PEO–Mb–AQ29D was thenexamined in ethanol, and a typical response is shown in Fig.7. The peak potential of PEO–Mb was approximately 130

mV more positive relative to that of native Mb in aqueoussolution. This shift is therefore attributed to solvation energydifferences around the exposed region of the heme moiety inPEO–Mb and Mb.

Immediately after characterization of the PEO–Mb–AQ29D/ethanol system aliquots of 0.1 M HCE in ethanolwere added to the cell. Fig. 8 shows that the addition of HCEproduced a catalytic response without a limiting currentregion, indicating that the diffusion of HCE to the Mb activesite was not the rate limiting factor at this sweep rate. Thecatalytic response was linearly dependent on the bulk con-centration of HCE as shown in Fig. 9.

AQ29D ionomer films contain both hydrophobic andhydrophilic micro-domains. As HCE is hydrophobic it isthought it will partition into the hydrophobic micro-domainsof the AQ29D film. The PEO modification of Mb may makeit partition into the hydrophobic micro-domains as well,thereby facilitating the reaction with HCE.

This work has shown that the PEO–Mb–AQ29D systemwill catalyse the reductive dehalogenation of HCE in ethanol,

M. Wright et al. / Electrochemistry Communications 1 (1999) 609–613 613


so it is hoped that this result can be extended to other halo-genated hydrocarbons that are soluble in ethanol but notwater. Myoglobin has been the focus of this initial commu-nication because it is commercially available and it can actin a similar manner to cytochrome P450 in a catalytic systemand has been used previously as an analogue to P450 [5],although it is less stereo- and regiospecific as a catalyst.

Recently it has been shown that cytochrome P450cam cancatalyse the reductive dehalogenation of a number of halo-genated hydrocarbons [7]. These experiments centred onchemical sources of electrons to drive the reduction. It hasalso been shown that the P450cam substrate binding site canbe altered by site-directed mutagenisis in order to change theenzyme specificity to other substrates [8,9]. Since the directelectrochemistry of this enzyme has been demonstrated [10]we now hope to build on this preliminary study by developingbiosensors specific for halogenated hydrocarbons based ongenetically engineered cytochrome P450 enzymes.

4. Note added in proof

Rusling and co-workers [11] have studied dehalogena-tions using Mb in surfactant films in aqueous solution.

Acknowledgements

M.J.H. would like to thank BG Technology for financialsupport and Steve Waugh for his continued inspiration. M.W.thanks the LEA for support.

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gradation 4 (1993) 39.[7] S. Li, L.P. Wackett, Biochemistry 32 (1993) 9355.[8] J.A. Stevenson, A.C.G. Westlake, C. Whittock, L.-L. Wong, J. Am.

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