the influence of non-phenolic mediators and phenolic co-substrates on the oxidation of 4-bromophenol...

The influence of non-phenolic mediators and phenolic co-substrates onthe oxidation of 4-bromophenol by lignin peroxidase

Gary Warda, Paula A. Belinkya, Yitzhak Hadarb, Itzhak Bilkisc, Carlos G. Dosoretzd,*aMIGAL-Galilee Technology Center, South Industrial Zone, Kiryat Shmona 10200, Israel

bDepartment of Plant Pathology & Microbiology, The Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew Universityof Jerusalem, Rehovot 76100, Israel

cInstitute of Biochemistry, Food Science and Nutrition, The Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew Universityof Jerusalem, Rehovot 76100, Israel

dDivision of Environmental and Water Resources Engineering, Faculty of Civil Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel

Abstract

Lignin peroxidase (LIP) catalyzed oxidation of 4-bromophenol (4-BP) resulted in the formation of dimers as evidenced by GC-MSanalysis. However, 4-BP was considered a poor substrate, since it exhibited a high Km value of 600.9 �M and during oxidation, LIP wassubjected not only to H2O2- but also product-dependent inactivation. Oxidation was enhanced by inclusion of gelatin, which suppressedproduct-dependent inactivation and by inclusion of the dimethoxylated non-phenols, veratrole (VO) and 3,4-dimethoxycinnamic acid(DMCA) which mediated 4-BP oxidation. The mediation role of VO and DMCA is attributed to their oxidation potential (OP) values in theaqueous phase (6.04 eV for VO and 6.03 eV for DMCA), which implies that once oxidized to their respective cation radicals, theypreferentially oxidize substrates of lower OP (5.12 eV for 4-BP phenolate ion). Inclusion of the mediators enabled complete 4-BP oxidationat high H2O2 concentrations, overcoming H2O2-dependent inactivation. When the reactive ferulic acid (FA) was included as a co-substrate,its oxidation preceded that of 4-BP, and since LIP was inactivated in the process, no oxidation of 4-BP was noticed. In the case ofhorseradish peroxidase (HRP), 4-BP oxidation was enhanced by inclusion of FA, even though oxidation of the latter preceded that of 4-BP.Additional studies indicated that this resulted from interaction of FA-oxidation products with 4-BP. In the presence of caffeic acid andcatechol, reduced oxidation of 4-BP was evident for both enzymes. In the presence of syringaldehyde, concomitant oxidation was evidentfor both enzymes, resulting in enhanced oxidation by HRP and moderate-to-slight enhancement by LIP. © 2002 Elsevier Science Inc. Allrights reserved.

1. Introduction

Lignin peroxidase (LIP), a heme-containing glycoproteinconsisting of several isoenzymes, participates in the extra-cellular depolymerization of lignin by the white-rot fungus,Phanerochaete chrysosporium [1]. The major role playedby LIP in lignin degradation is attributed in part to its redoxpotential, reported to be higher than that of any other per-oxidase [2,3]. This enables LIP to catalyze reactions notnormally associated with other peroxidases, in particular theoxidation of non-phenolic aromatic substrates. It has beenreported that LIP can oxidize aromatic compounds withcalculated ionization potential (IP) values of up to 9.0 eV [4].

The wide substrate range of LIP has striking implicationswhen considering emerging applications for peroxidases in

bioremediation and catalysis of difficult chemical transfor-mations [5–6]. One such application includes the catalyticremoval of phenolic and other aromatic compounds fromwastewater [7–11]. Phenols are oxidized by peroxidases togenerate phenoxyl radicals, which couple to form dimeric,oligomeric and polymeric products. The increase in molec-ular weight results in precipitation and a decrease in reac-tivity of the products, which is apparently accompanied bya detoxification effect [10]. The method presents a possiblealternative for the high-rate treatment of industrial waste-water when conventional methods may be ineffective due tothe nature of the wastewater stream [8]. Halogenated phe-nols, which constitute a significant category of pollutantsfrom a wide variety of industries, would be primary targetsin peroxidase catalyzed treatment of wastewater. However,the presence of electron withdrawing groups on the phenolsis expected to increase their IP values significantly, makingthem relatively poor substrates for LIP. Furthermore, LIP isexpected to be prone to H2O2- and product-dependent in-

* Corresponding author. Tel.: �972-4-829-4962; fax: �972-4-822-8898.

E-mail address: [email protected] (C.G. Dosoretz).

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activation during their oxidation, which would significantlydecrease operational stability. LIP is very susceptible toH2O2-dependent inactivation in comparison to other peroxi-dases due to the high reactivity of LIP compound II (LIPII)with H2O2 yielding LIP compound III (LIPIII) [12–15].Failure to revert LIPIII to the native ferric state results in itsinactivation, a common phenomenon witnessed during theoxidation of phenols [16–18]. Product-dependent inactiva-tion during the oxidation of phenols is attributed to theirreversible binding of phenoxyl radical intermediates orproducts to the enzyme [7,8,18–21].

Various studies have shown that such limitations can beovercome to a certain degree. Firstly, it has been shown thatveratryl alcohol (VA), which is produced de novo by ligni-nolytic cultures of P. chrysoporium, can act as a redoxmediator for LIP-catalyzed oxidation. VA is oxidized byLIP to the VA radical cation, which in turn acts as a charge-transfer mediator, oxidizing the substrate [18,22–27]. Oxida-tion of poor phenolic substrates is enhanced in the presence ofVA, which is attributed to its ability to act as a redox mediatorand in part due to its ability to revert LIPIII to the native ferricstate, thus protecting LIP from H2O2-dependent inactivation[12,14,15]. Secondly, additives such as gelatin and polyethyl-ene glycol have been shown to suppress product dependentinactivation of horseradish peroxidase (HRP) [21] and morerecently LIP [18], presumably by competitively binding to thephenoxyl radicals or polymerized phenol. Thirdly, it has alsobeen shown with other peroxidases but not LIP, that co-sub-strates that react readily with the enzyme enhance the oxidationof less reactive phenols [7,28,29]. The most plausible mecha-nism for the enhancement of substrate removal is thought to bea secondary chemical reaction between less reactive substratesand intermediates or products formed during oxidation of re-active substrates, resulting in the formation of mixed polymers[29]. The enhancement of oxidation of less reactive substrateshas been shown only to occur in the presence of those reactivesubstrates that follow a similar mechanism of oxidation [28].

In this work, we studied the in vitro LIP-catalyzed oxi-dation of 4-bromophenol (4-BP), acknowledged as a toxichalogenated phenol. Our aim was to study the effect ofnovel non-phenolic mediators, phenolic co-substrates andother additives on the extent of 4-BP oxidation in order tofurther understand the reaction mechanisms and in order toconclude which measures are most suitable in order toenhance oxidation. Studies were also conducted on 2,4-dibromophenol (2,4-DBP) and the performance of LIP wascompared to that of HRP.

2. Materials and Methods

2.1. Chemicals

H2O2 (30% v/v solution) and ferulic acid (FA) were bothobtained from Sigma. Veratrole (3,4-dimethoxybenzene,VO) was obtained from Fluka and 3,4-dimethoxycinnamic

acid (DMCA), 4-BP and syringaldehyde (SYR) from Al-drich. The benzofuran form of �-5�-diferulic acid and thedilactone form of �,��-diferulic acid, both of which areproducts of LIP-catalyzed oxidation of FA were isolated aspreviously described [30]. The concentration of stock solu-tions of H2O2 was determined at 240 nm using an extinctioncoefficient of 39.4 M�1 cm�1. Stock solutions of 4-BP,2,4-DBP, FA, SYR, VO and DMCA were made in 95%ethanol and checked regularly using calculated extinctioncoefficients of 1,300 M�1 cm�1 at 280 nm, 1,700 M�1

cm�1 at 286 nm, 14,700 M�1 cm�1 at 320 nm, 2,900 M�1

cm�1 at 270 nm, 2,100 M�1 cm�1 at 273 nm, and 15,900M�1 cm�1 at 314 nm, respectively. All stock solutions were100 mM, except DMCA, which was 10 mM.

2.2. Enzymes

LIP isoenzyme H1 was produced from high-nitrogencultures of P. chrysosporium Burds BKM-F-1767 [31] andpurified as described previously [18]. The purified enzymehad an RZ (A409/A280) value � 4.0. LIP concentration wasdetermined at 409 nm using an extinction coefficient of 169mM�1 cm�1 [32]. LIP activity (U/liter) was assayed ac-cording to Tien and Kirk [33] and the catalytic activity ofthe stock enzyme solution was calculated to be 1.96 U pernmol heme protein. The enzyme was extensively dialyzedagainst double-distilled water before use.

HRP type I with an RZ (A409/A280) of 1.0 and an activityof 78 U/mg solid was purchased from Sigma. One unit ofactivity forms 1 milligram of purpurogallin from pyrogallolin 20 s at pH 6.0 and 20°C, monitored spectrophotometri-cally at 420 nm.

2.3. Enzyme reactions

All LIP reactions were carried out in 50 mM sodiumtartrate buffer, pH 3.5 at 25°C [18]. All HRP reactions werecarried out in 200 mM sodium acetate buffer, pH 5.5 [28].Enzyme, reducing substrate and H2O2 concentrations alongwith other additions are given in the figure legends.

2.3.1. Steady-state kineticsThe steady-state kinetic constants Km and kcat were cal-

culated from initial velocity studies, in which 300 �M H2O2

was added to mixtures containing 0.2 �M LIP, substrate(0–2000 �M) and 50 mM tartrate buffer, pH 3.5. Thereactions were stopped after 10 s by adding an equal volumeof acetonitrile to inactivate the enzyme and remaining sub-strate was then quantified by HPLC. The amount of sub-strate consumed in 10 s (calculated from blanks withoutH2O2 and authentic compounds) for different initial sub-strate concentrations was used to calculate Km and kcat.Three replicates of both blanks and reactions were analyzed.

491G. Ward et al. / Enzyme and Microbial Technology 30 (2002) 490–498

2.3.2. Substrate consumption studiesConsumption studies were conducted using 1 �M LIP,

1000 �M substrate and 0–100 �M H2O2. Remaining sub-strate was determined by HPLC analysis 1 h after thereactions were initiated.

2.3.3. Residual LIP activityResidual LIP activity was tested on 100 �l samples taken

from reactions that were incubated for 5 min at 25°C con-taining 1 �M LIP, 500 �M substrate and 250 �M H2O2.Gelatin, when added was 0.4 mg/ml. Residual LIP activitywas assayed by monitoring the oxidation of VA at 310 nmin 1 ml mixtures consisting of 2 mM VA, 0.4 mM H2O2 and50 mM sodium tartrate buffer, pH 2.5 [33]. A 500 �l aliquotwas also withdrawn from reactions and immediatelyadded to an equal volume of acetonitrile to quench thereaction. Remaining substrate was then determined byHPLC.

2.4. HPLC analysis

HPLC analysis was conducted on a Hewlett PackardHPLC (HP1100 series) using a Lichrospher 100 RP-C18column (25 cm � 5 mm; i.d., 5 �m; Merck) with monitor-ing at 280 nm. Elution was performed using a gradientsystem that increased the relative amount of methanol andacetonitrile in the mobile phase consisting of 1 mM triflu-oroacetic acid as previously described [30]. The flow ratewas maintained at 1 ml/min. Concentrations were deter-mined using authentic compounds.

2.5. GC-MS analysis

Reactions containing 1000 �M substrate were oxidizedby 1 �M LIP and a total of 1000 �M H2O2, which wasadded in aliquots of 250 �M at 1-min intervals. After 1 h,reactions were frozen at �70°C and freeze-dried. Driedproducts were silylated in 200 �l dioxane with 200 �lN,O-bis (trimethylsilyl)-acetamide (BSA) for 30 min at60°C and then analyzed by GC-MS as reported previously[30].

2.6. Quantum chemical calculations

The semi-empirical AM1 quantum chemical method wasused for calculating the optimal geometries and heat offormation of the substrates and their radical cations and freeradicals in an aqueous environment. The difference in theheat of formation of the substrates and radicals correspondsto the oxidation potential (OP) in an aqueous environment.All the calculations were performed with the Gaussian 94[34] and Spartan 5.1 programs.

3. Results

3.1. Direct oxidation of 4-BP by LIP

The oxidation of both 4-BP and 2,4-DBP by LIP fol-lowed Michaelis Menten kinetics with Km values of 600.9�M and 387.3 �M, respectively. The kcat values were 41.2s�1 for 4-BP and 42.7 s�1 for 2,4-DBP. The OP values ofthe phenolate ions of 4-BP and 2,4-DBP in the aqueousphase were calculated as being 5.12 eV and 5.26 eV, re-spectively.

Peroxidase-catalyzed oxidation of phenols results in theformation of phenoxyl radicals, which have a tendency topolymerize. Upon GC-MS analysis of 4-BP oxidation prod-ucts after silylation, the major component of the molecularion cluster was characterized by isotopic peaks M� � 486(45%), M � 2 (100%) and M � 4 (50%) with an intensityratio �1:2:1, typical of dibromo organic compounds, indi-cating the formation of the 2,2-dimer of 4-BP. According tothe results of GC-MS analysis of 2,4-DBP oxidation prod-ucts after silylation, the major component was ascribed tothe disilylated 6,6-dimer of 2,4-DBP: the molecular ioncluster was characterized by isotopic peaks M� � 642(4%), M � 2 (20%), M � 4 (25%), M � 6 (16%), M � 8(5%) with an intensity ratio �1:4:6:4:1, typical for tetra-bromo organic compounds The structures of the identifieddimers are given (Fig. 1). Further analysis of oxidationproducts by gel permeation showed peaks with molecularweights of �500 and 700 Da for 4-BP and 750 and 1000 Dafor 2,4-DBP, indicating that trimers and tetramers may alsohave been formed during oxidation.

Substrate consumption studies indicated that as much as1.76 mol 4-BP and 1.89 mol 2,4-DBP were consumed permol of H2O2, by LIP. However, LIP-catalyzed oxidationwas very sensitive to H2O2 (Fig. 2). Under the reactionconditions shown, oxidation peaked at 100 �M H2O2, with132 �M 4-BP being oxidized. Further increases in H2O2

resulted in a progressive decrease in the amount of 4-BPoxidized. This trend typifies H2O2-dependent inactivationand spectral analysis of reaction mixtures at time intervalsrevealed the gradual appearance of LIPIII.

Fig. 1. Dimers formed during oxidation of 4-BP and 2,4-DBP by LIP. Thestructures were determined after silylation by GC-MS analysis.

492 G. Ward et al. / Enzyme and Microbial Technology 30 (2002) 490–498

3.2. Oxidation of 4-BP in the presence of dimethoxylatednon-phenolic mediators

VA has been shown to preferentially mediate and en-hance the oxidation of phenolic substrates [18,22–27] andalso prevent H2O2-dependent inactivation by effectivelyreverting LIPIII to the native state [12,14–15]. Results ofour own indicate that other dimethoxylated non-phenolicsubstrates, namely VO and DMCA (calculated OP values of6.04 and 6.03 eV, respectively) are also capable of mediat-ing oxidation of phenols by LIP. Inclusion of VO resulted ina mediation phenomenon, with 4-BP oxidation precedingthat of VO, even though the concentration of the latter was15 times higher (Fig. 3). The presence of VO resulted in

complete 4-BP oxidation at H2O2 concentrations that in itsabsence were characterized by incomplete oxidation (Fig.3). The same phenomenon was noticed when 4-BP wasreplaced with 2,4-DBP.

3.3. Oxidation of 4-BP in the presence of gelatin

After incubation with 500 �M 4-BP and 250 �M H2O2

for 5 min, 1 �M LIP had oxidized 351.3 �M 4-BP, yet only21.3% activity was recovered (Table 1). Inclusion of gelatinin the reaction mixtures resulted in 366.4 �M 4-BP beingoxidized and a recovery of 85.7% enzyme activity. A sim-ilar phenomenon was observed for both 2,4-DBP and FA(Table 1). Gelatin did not prevent H2O2-dependent inacti-vation since its inclusion with H2O2 in the absence ofreducing substrate did not preserve enzyme activity. Sincein addition, a previous report has shown that this type ofinactivation occurred even at low H2O2 concentrations andit cannot be avoided by inclusion of mediators, it appears tobe independent of H2O2 [18]. Non-phenolic substrates donot appear to subject LIP to this type of inactivation, sinceinclusion of gelatin had no influence on residual activity(Table 1).

3.4. Oxidation of 4-BP in the presence of the co-substrateFA

The effect of the inclusion of FA as a co-substrate on theoxidation of 4-BP was studied. FA was preferentially oxi-dized by LIP and competitively inhibited the oxidation of

Fig. 2. Effect of H2O2 on the oxidation of 4-BP by LIP. Reactions consistedof 0.2 �M LIP, 300 �M 4-BP, 50 mM tartrate buffer, pH 3.5 and varyingconcentrations of H2O2. Reactions were allowed to run to completion andremaining substrate was determined by HPLC, with monitoring at 280 nmat least 1 h after onset of the reaction.

Fig. 3. LIP-catalyzed oxidation of 4-BP in the presence of VO. 4-BPoxidation in the presence (F) and absence of VO (E), VO oxidation in thepresence of 4-BP (■ ). Reaction mixtures contained 0.2 �M LIP, 100 �M4-BP, 50 mM tartrate buffer, pH 3.5, 400 �M H2O2 and when added, 1500�M VO. The reactions were stopped at designated times by addition of anequal volume of acetonitrile and remaining substrate determined by HPLC,with monitoring at 280 nm.

Table 1Residual LIP activity in different reaction mixtures1

ResidualActivity(% of blank)

Substrateconsumed(�M)

LIP � H2O2 2.9 � 0.2 naLIP � H2O2 � gelatin 3.6 � 0.9 naLIP � 4-BP � H2O2 21.3 � 0.7 351.3 � 1.3LIP � 4-BP � H2O2 � gelatin 85.7 � 4.8 366.4 � 0.8LIP � 2,4-DBP � H2O2 3.4 � 0.4 439.2 � 15.2LIP � 2,4-DBP � H2O2 � gelatin 87.6 � 0.8 479.4 � 0.9LIP � FA � H2O2 10.6 � 6.2 383.5 � 1.9LIP � FA � H2O2 � gelatin 73.7 � 2.6 390.1 � 1.4LIP � VA � H2O2 82.5 � 0.8 332.5 � 0.2*LIP � VA � H2O2 � gelatin 81.1 � 3.8 302.6 � 1.5*LIP � VO � H2O2 68.4 � 0.6 144.3 � 0.7LIP � VO � H2O2 � gelatin 68.1 � 0.5 128.4 � 4.3LIP � DMCA � H2O2 68.2 � 1.2 291.8 � 26.7LIP � DMCA � H2O2 � gelatin 68.5 � 1.1 237.2 � 5.6

1 Residual LIP activity was tested on 100 �l samples taken from reac-tions that contained 1 �M LIP, 500 �M substrate, 250 �M H2O2 and 50mM sodium tartrate buffer, pH 3.5 that were incubated for 5 min at 25°C.Gelatin, when added was 0.4 mg/ml. Residual activity is presented as apercentage of the respective blank, which was either LIP alone or LIP �gelatin. Results are presented as the average of 3 replicates � SD.

na: not applicable.*: veratryl aldehyde produced.


4-BP (Fig. 4a). Since oxidation of the more reactive FA(OP � 4.78 eV in aqueous phase) preceded that of 4-BP,this shows that there was no direct interaction between thetwo. Phenoxyl radicals produced during the oxidation of FAonly coupled with like molecules as witnessed by the pres-ence of peaks corresponding to previously identified FAproducts [30]. A similar phenomenon was witnessed when4-BP was replaced with 2,4-DBP.

In the absence of 4-BP, incomplete oxidation of FA wasevident when the ratio FA:H2O2 was lower than 2:1, theminimum ratio required to avoid excess H2O2 [18] (Fig. 4b).Inclusion of 1500 �M 4-BP in the reaction mixture in-creased the ratio phenol:H2O2 and resulted in completeoxidation of FA under otherwise identical conditions. Insuch case, even less oxidation of 4-BP was evident than inthe absence of FA. This possibly indicates that 4-BP pro-vided limited protection to the enzyme from H2O2-inacti-vation, enabling complete oxidation of FA.

Similar to reactions for LIP, FA oxidation by HRP pre-ceded that of 4-BP, indicating that it was preferentiallyoxidized, thus inhibiting 4-BP oxidation (Fig. 5). However,

two major differences were evident. Firstly, HRP-catalyzedoxidation of FA occurred much faster than for LIP, all of theFA being oxidized in the first 10 s. Secondly, the extent of4-BP oxidation by HRP was enhanced in the presence ofFA. Although the initial rate of 4-BP oxidation was higherin the absence of FA, 4-BP oxidation ceased almost com-pletely after 60 s. On the other hand, in the presence of FA,4-BP oxidation took place at a lower initial rate, but con-tinued at a steady rate, and after 300s of reaction, more 4-BPhad been oxidized in the presence, than in the absence ofFA. A similar phenomenon was witnessed for 2,4-DBP. FAitself did not undergo interaction with 4-BP, since it haddisappeared before onset of 4-BP oxidation.

HRP-catalyzed oxidation of 4-BP was enhanced by in-clusion of gelatin in reaction mixtures as for LIP (Fig. 6).Interestingly, inclusion of gelatin in reactions containing4-BP and FA did not further enhance oxidation.

In order to study whether the enhancement of 4-BPoxidation in the presence of FA by HRP was due to FA-oxidation products, the latter were isolated and added toreactions containing HRP and 4-BP, to which H2O2 wasthen added (Table 2). In the presence of �-5�-diferulic acidor �-��-diferulic acid, 300 �M 4-BP was completely oxi-dized by 0.15 U HRP and 500 �M H2O2, whereas in theabsence, less than 50% of the 4-BP was oxidized. Resultsfor LIP, also given in Table 2, indicate that less 4-BP wasoxidized in the presence than in the absence of the FA-oxidation products, indicating competitive inhibition as no-ticed for FA itself.

3.5. Oxidation of 4-BP in the presence of otherco-substrates

Caffeic acid (CAFF) (OP � 4.83), catechol (CAT)(OP � 4.83), 2,6-dimethoxyphenol (2,6-DMP) (OP �

Fig. 4. LIP-catalyzed oxidation of 4-BP in the presence of FA. A. 4-BP(F), FA (■ ). Reaction mixtures consisted of 0.2 �M LIP, 300 �M 4-BP,300 �M FA, 50 mM tartrate buffer, pH 3.5 and varying amounts of H2O2.

Reactions were allowed to run to completion and analyzed as for Fig. 2. B.FA oxidation in the presence (■ ) and absence (�) of 4-BP, 4-BP oxidationin the presence (F) and absence (E) of FA. Reaction mixtures consisted of0.2 �M LIP, 50 mM tartrate buffer, pH 3.5, 300 �M H2O2 and when added1500 �M 4-BP and 50 �M FA, together or alone. The reactions werestopped at designated times and analyzed as for Fig. 3.

Fig. 5. HRP-catalyzed oxidation of 4-BP in the presence of FA. 4-BPoxidation in the presence (F) or absence (E) of FA (■ ). The reactionsconsisted of 0.15 U/ml HRP, 300 �M 4-BP, 200 mM acetate buffer, pH5.5, 500 �M H2O2 and when added, 300 �M FA. Reactions were stoppedat designated times and analyzed as for Fig. 3.


4.66), and guaiacol (GUA) (OP � 4.79) all competitivelyinhibited LIP-catalyzed oxidation of 4-BP in a similar man-ner to FA (data not shown). However, in the presence ofSYR (OP � 4.92), both substrates were concomitantly ox-idized, and the extent of 4-BP oxidation was dependent onthe concentration of SYR included (Fig. 7). Increasing theconcentration of SYR up to 800 �M resulted in enhanced4-BP oxidation. However, thereafter a decrease in 4-BPoxidation was noticed. The influence of FA and VO concen-trations on the oxidation of 4-BP are shown to highlight thethree different phenomena witnessed when different substrateswere added to reactions containing LIP and 4-BP (Fig. 7).

HRP catalyzed oxidation of 4-BP was also enhanced inthe presence of SYR (Fig. 8). Concomitant oxidation of

both substrates took place as for LIP. In the same reactions,more SYR was oxidized in the presence, than in the absenceof 4-BP. However, although enhanced 4-BP oxidation byHRP was noticed in the presence of both SYR and FA, 4-BPoxidation was reduced in the presence of CAFF and CAT.

4. Discussion

Both 4-BP and 2,4-DBP have a low affinity and can beconsidered poor substrates for LIP, since they exhibit rela-tively high Km values in comparison with other phenolicsubstrates.1 Electron-withdrawing substituents such as bro-mine on the aromatic ring of phenols are expected to result

Fig. 6. HRP-catalyzed oxidation of 4-BP in the presence of FA and gelatin.Oxidation of 4-BP alone (E), in the presence of gelatin (■ ), in the presenceof FA (F), and in the presence of FA and gelatin (�).The reactionsconsisted of 0.15 U/ml HRP, 300 �M 4-BP, 500 �M H2O2 and 200 mMacetate buffer, pH 5.5 and when added 300 �M FA and 0.4 mg/liter gelatin.Reactions were stopped and analyzed as for Fig. 3.

Table 2Influence of FA oxidation-products on the oxidation of 4-BP by both HRP and LIP1

Enzyme Substrate 0 �M H2O2 500 �M H2O2

4-BP remaining(�M)

FA-product remaining(area of peak at 280 nm)

4-BP remaining(�M)

FA-product remaining(area of peak at 280 nm)

0.15 U/ml HRP2 300 �M 4-BP 296.6 � 2.5 165.0 � 19.5300 �M 4-BP � �-5�-diferulic acid 335.3 � 62.9 7131.7 � 436.2 nd nd300 �M 4-BP � �-��-diferulic acid 311.4 � 6.9 1317.4 � 65.8 nd nd�-5�-diferulic acid 6889.7 nd�-��-diferulic acid 1868.9 nd

0.5 �M LIP3 300 �M 4-BP 285.6 � 3.0 156.6 � 12.8300 �M 4-BP � �-5�-diferulic acid 314.4 � 5.3 5064.6 � 35.1 250.4 � 2.6 1245.1 � 110.1300 �M 4-BP � �-��-diferulic acid 302.6 � 0.8 1417.2 � 50.2 223.9 � 1.1 324.5�-5�-diferulic acid 4881.6 � 132.2 1876.7 � 143.6�-��-diferulic acid 1475.7 � 175.8 307.2

1 Reactions were allowed to reach completion and after at least 1 hour remaining substrate was determined by HPLC analysis. FA oxidation products wereproduced and isolated according to Ward et al. [30]. Results are presented as the average of 3 replicates � SD.

2 HRP reactions were carried out in 200 mM sodium acetate buffer, pH 5.5.3 LIP reactions were carried out in 50 mM sodium tartrate buffer, pH 3.5 at 25°C.nd: not detectable.

Fig. 7. Comparison of the effect of mediators and co-substrates on theextent of oxidation of 4-BP by LIP. 4-BP oxidation in the presence of VO(■ ), in the presence of FA (E) and in the presence of SYR (F). Reactionsconsisted of 0.5 �M LIP, 300 �M 4-BP, 50 mM tartrate buffer, pH 3.5 and500 �M H2O2 in the presence of varying concentrations of mediator orco-substrates. Reactions were allowed to run to completion and analyzed asfor Fig. 2.


in a corresponding increase in the OP, making it increas-ingly difficult to remove electrons. The OP of the phenolateions of 4-BP and 2,4-DBP in the aqueous phase were cal-culated as being 5.12 eV and 5.26 eV, respectively, valuesthat are relatively high in comparison with other phenols.1

The bromophenols were oxidized by LIP to phenoxylradicals, which coupled resulting in the formation of highermolecular weight polymers. Although GC-MS analysis con-firmed the presence of dimers, and gel permeation analysissuggested that trimers and tetramers were also formed, themaximum extent of polymerization is uncertain. This prob-ably depends on the OP values of products, which determinewhether or not they can be further oxidized, and enzymestability and H2O2, which determine if the maximum degreeof polymerization is attained.

During oxidation of the bromophenols, LIP was sub-jected to the well-known H2O2-dependent inactivation [16–18] and product-dependent inactivation [18]. The significantdecrease in LIP activity resulting from product-dependentinactivation during oxidation of 4-BP and 2,4-DBP could besuppressed by inclusion of gelatin. Studies on other peroxi-dases suggest that additives such as gelatin and polyethyl-ene glycol can suppress the inactivation by competitivelybinding to the phenoxyl radicals or polymerized phenol[21]. Gelatin and other additives may also protect the en-zyme by temporarily blocking hydrogen bonding sitesthereby preventing hydrogen bonding between the hydroxylgroups of the phenolic products and the enzyme.

On account of the low reactivity of the bromophenolsand the low operational stability of LIP, attempts were madeto enhance oxidation by including redox mediators. VA isknown to mediate oxidation of phenolic substrates resultingin enhanced oxidation [18,22–27]. Our results indicate thatDMCA and VO, which like VA are dimethoxylated non-phenolic substrates, were also capable of mediating oxida-tion of both 4-BP and 2,4-DBP. The ability of VO and

DMCA to mediate oxidation is attributed in part to differ-ences in OP values. In aqueous phase, the OP of VO andDMCA were calculated to be 6.04 and 6.03 eV, respec-tively, which are higher than the OP of the phenolate ions of4-BP and 2,4-DBP, calculated at 5.12 eV and 5.26 eV. Thisimplies that even though at the concentrations used, VO andDMCA saturated LIP, once oxidized they were reduced bythe bromophenol substrates. As a result the oxidation of thebromophenols preceded that of their non-phenolic counter-parts and only when all of the bromophenol substrate hadbeen oxidized did onset of VO or DMCA oxidation begin.Enhanced oxidation of the bromophenols was witnessed asa result. Since VO and DMCA are able to revert LIPIII tothe native state, albeit less efficiently than VA1, this mayalso explain why H2O2-dependent inactivation was sup-pressed by their inclusion. Since in addition, the fungalsecondary metabolite, 2-chloro-1,4-dimethoxybenzene wasshown to act as redox mediator for LIP catalyzed reactions[35], this suggests the possible physiological role of otherfungal metabolites besides VA as redox mediators.

Further attempts to enhance oxidation were made by theinclusion of reactive phenolic compounds as co-substrates.Oxidation of less reactive substrates by oxidases and per-oxidases has been reported to be enhanced in the presenceof reactive substrates that follow a similar mechanism ofoxidation [7,28,29]. This effect may be explained by anincrease in the yield of free radicals, which results in theformation of co-polymers, thus displacing the reaction.Nevertheless, neither enhanced oxidation of 4-BP nor 2,4-DBP by LIP was observed when FA was included in thereaction mixtures and FA oxidation preceded that of thebromophenols. The OP value for FA phenolate ion in aque-ous phase was calculated as 4.78 eV, which is lower thanthe respective values for both 4-BP and 2,4-DBP. FA istherefore a more reactive substrate and exhibits a lower Km

of 116.8 �M and a kcat of 41.7 s�1 [18]. Therefore it is notsurprising that FA was preferentially oxidized. Since LIPunderwent inactivation during the oxidation of FA, no 4-BPoxidation was witnessed under the reaction conditions em-ployed.

When HRP was employed instead of LIP, enhancedoxidation of both 4-BP and 2,4-DBP was witnessed in thepresence of FA. These findings comply with other reportsthat showed that FA enhanced oxidation of chlorinatedphenols by HRP [28]. Since as for LIP, HRP-catalyzedoxidation of FA preceded 4-BP oxidation, enhancementprobably results from interaction of FA products with 4-BP.This assumption is strengthened by the fact that when 4-BPwas oxidized by HRP in the presence of the FA-oxidationproducts �-5�-diferulic acid and �-��-diferulic acid, en-hanced oxidation was again witnessed. This was not thecase for LIP.

The behavior witnessed when other co-substrates wereincluded was not always similar to that for FA. For bothreduced oxidation of 4-BP was witnessed in the presence ofCAFF and CAT both of which like FA possess considerably

Fig. 8. Influence of various co-substrates on the extent of HRP-catalyzedoxidation of 4-BP. 4-BP (■ ), co-substrate (�). Reactions consisted of 0.15U/ml HRP, 300 �M 4-BP, 500 �M H2O2, 200 mM acetate buffer, pH 5.5and when added 300 �M co-substrate. Reactions were allowed to reachcompletion and analyzed as for Fig. 2.


lower OP values. In the presence of SYR, concomitantoxidation of substrates was evident for both enzymes, re-sulting in enhanced oxidation by HRP and moderate toslight enhancement by LIP. The calculated OP value forSYR is more similar to that of 4-BP than the others. Dif-ferences in OP values may help understand the differentialbehavior witnessed, however this may also be a result ofdifferent mechanisms as suggested by others [28].

In conclusion, four different phenomena were evidentwhen additional substrates or additives were included inreaction mixtures containing 4-BP or 2,4-DBP and LIP: (i)Enhanced oxidation in the presence of mediators such asVA, VO and DMCA; (ii) Competitive inhibition in thepresence of substrates such as FA, CAFF, CAT, GUA and2,6-DMP; (iii) Partial enhancement in the presence of co-substrates such as SYR; and (iv) Partial-to complete sup-pression of product-dependent inactivation by inclusion ofgelatin.

Notes

1. Ward G, Bilkis I, Hadar Y, and Dosoretz CG, un-published data.

Acknowledgments

This research was supported by The Israel Science Foun-dation founded by The Israel Academy of Science andHumanities (Grant No 655/99–2) and the Fund for thePromotion of Research at the Technion.

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the influence of non-phenolic mediators and phenolic co-substrates on the oxidation of 4-bromophenol...

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