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Spectrophotometric assay for horseradish peroxidase activity based on pyrocatechol–aniline coupling hydrogen donor
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 362 (2007) 38–43 www.elsevier.com/locate/yabio
Spectrophotometric assay for horseradish peroxidase activity based on pyrocatechol–aniline coupling hydrogen donor A. Molaei Rad a, H. Ghourchian a
a,*
, A.A. Moosavi-Movahedi a, J. Hong a, K. Nazari
b
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran b Research Institute of Petroleum Industry, Tehran, Iran Received 27 August 2006 Available online 20 December 2006
Abstract The hydrogen donor couples pyrocatechol–aniline and phenol–aminoantipyrine in the presence of hydrogen peroxide were compared as chromogens for horseradish peroxidase (HRP) assay. UV–Visible spectroscopy and high-performance liquid chromatography analysis indicated that during the HRP biocatalytic process, pyrocatechol–aniline was converted to a pink-colored reagent with a kmax of 510 nm, which was used in the assay of HRP activity. Electrochemical studies revealed adequate electron transfer ability for this color reagent to serve as a proper mediator for HRP also. Using pyrocatechol–aniline a higher sensitivity and lower detection limit was obtained relative to those of the phenol–aminoantipyrine couple, which is commonly used for HRP assay. A relative standard deviation of 2.9% was obtained for 20 HRP activity measurements, indicating a satisfactory reproducibility for this method. In addition, kinetic parameters of Km (12.5 mM) and Vmax (12.2 mM min1 mg1) were calculated for pyrocatechol–aniline. Regarding the superiority of pyrocatechol–aniline, this couple is suggested to be a better hydrogen donor for the HRP spectrophotometric assay. 2006 Elsevier Inc. All rights reserved. Keywords: Horseradish peroxidase; Hydrogen donor; Pyrocatechol; Aniline; Enzyme assay
Horseradish peroxidase (EC 1.11.1.7; donor-H2O2 oxidoreductase) (HRP)1 is one of the heme peroxidases, which catalyze a variety of oxidative transformations of organic and inorganic substrates by hydrogen peroxide or alkyl peroxides [1–7]. Furthermore, most phenolic compounds are subject to oxidative coupling and the reaction can be catalyzed by certain naturally occurring extracellular enzymes and peroxidases [8]. The activity of these peroxidases has traditionally been expressed in units based upon the rate of oxidation of pyrogallol introduced by Willstalter and Stoll in 1917. The simplicity, rapidity, facile and *
Corresponding author. Fax: +98 21 66404680. E-mail address: [email protected] (H. Ghourchian). 1 Abbreviations used: HRP, horseradish peroxidase; HPLC, high-performance liquid chromatography; ESR, electron spin resonance; LC, liquid chromatography; ABTS, 2,2-azino-bis(3-ethylbenzothiazolin-6-sulfonate); TMPOH, 2,2,6,6-tetramethyl piperidone-oxime hydrochloride; PBS, phosphate buffer solution; CV, cyclic voltammetry; R.S.D., relative standard deviation. 0003-2697/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.11.035
inexpensive properties of spectrophotometry have made it a popular method to determine activity of HRP [9], therefore more detailed studies by using this technique have been presented since 1954 [10]. A wide variety of hydrogen donors (e.g., caffeic acid and 2,2-azino-bis(3-ethylbenzothiazolin-6-sulfonate)) have been utilized in peroxidase assay systems including methods using potentially carcinogenic and mutagenic materials [11,12]. In addition, an improved spectrophotometric assay has been adopted using 4-aminoantipyrine and phenol as hydrogen donors, although phenol itself is carcinogenic and must be handled with extra precautions. In this method, the reaction rate is determined by measuring the increase in absorbance caused by the formation of a dye complex resulting from the decomposition of hydrogen peroxide [13]. In the present study, we have proposed the pyrocatechol– aniline couple as hydrogen donor for horseradish peroxidase assay and developed an improved spectrophotometric method for the determination of HRP activity.
Spectrophotometric assay for horseradish peroxidase activity / A. Molaei Rad et al. / Anal. Biochem. 362 (2007) 38–43
Materials and methods Reagents Horseradish peroxidase types II with a purity index (RZ) of 2.0, hydrogen peroxide, and finer alumina powder were obtained from Sigma. Phenol, 4-aminoantipyrine, sodium phosphate, pyrocatechol (1,2-dihydroxybezene), 2,2,6, 6-tetramethyl piperidone-oxime hydrochloride (TMPOH), aniline, and acetonitrile (99.8%) were obtained from Merck. All solutions were prepared in double-distilled deionized water (Barnstead Nanopure D4742, resistance 18.3 MX). HRP activity assay The activity of the enzyme was determined colorimetrically using a UV–Vis spectrophotometer (Cary 100 Bio, Varian, Australia), equipped with a temperature controller. A mixture of pyrocatechol (170 mM) and aniline (2.5 mM) was prepared in phosphate buffer solution (PBS) 0.2 M, pH 7.0. To each blank and sample cuvettes, 450 ll of the above mixture solution and 500 ll of hydrogen peroxide (1.7 mM) were pipetted. The spectrophotometer was adjusted to 510 nm. To achieve a temperature equilibration both mixtures in cuvettes were incubated in spectrophotometer at 25 C for 3–4 min. Then, 50 ll of diluted HRP and 50 ll PBS were added to the sample and blank cuvettes, respectively. Increase in absorbance was recorded for 4–5 min. The concentration of HRP should be so diluted that a certain reaction rate (usually, DA/min = 0.02–0.04) could be obtained. The reaction rate was determined by measuring the increase in absorbance resulting from the decomposition of hydrogen peroxide and formation of the colored product of 4-(phenylamino)benzene-1,2-diol (Scheme 1b). Prior to each assay, the concentration of hydrogen peroxide was determined based on its absorbance at 240 nm and extinction coefficient (e240 = 43.6 cm1 M1) [14,15]. The activity of HRP was calculated based on Eq. (1), units=mg ¼
DA= min ; e mgðenzymeÞ=mlðreaction mixtureÞ
ð1Þ
where e (extinction coefficient) for 4-(phenylamino)benzene-1,2-diol is 5 mM1 cm1. To evaluate the performance of this method, the obtained results were compared with those acquired from the conventional method depicted in the Worthington manual [13]. To estimate the capability of other hydrogen donor couples in HRP assay, the same procedure was used except that the spectrophotometer was adjusted to 360 nm for pyrocatechol–aminoantipyrine and to 310 nm for pyrocatechol–phenol, pyrocatechol–TMPOH, and phenol– aniline. HPLC analysis High-performance liquid chromatography separation was performed on a Chromatopac Shimadzu C-R4A, Sys-
39
tem controller SCL-6A, equipped with a UV–Vis spectrophotometer detector Model SPD-6AV, liquid chromatograph LC-6A, and a LiChrospher 100 column, RP-18 (5-lm particle size)-LiChroCART 250-4. A solution of PBS (0.2 M, pH 7) with total volume of 1 ml containing H2O2(1.7 mM), pyrocatechol (170 mM) and aniline (2.5 mM) was prepared at 25 C. To avoid column contamination by the enzyme, the HRP solution was kept in a dialysis bag, cutoff 10 kDa, while it was exposed to the reaction mixture. A pink-colored component was formed gradually. Then, 20 ll of the mixture was directly injected into the column. A mixture of acetonitrile–water with a flow rate of 1.0 ml/min was used as the mobile phase with a ratio of 10/90 (v/v) at starting point. The ratio was gradually changed to 90/10 (v/v) within a period of 60 min. Chromatograms for the determination of pyrocatechol, aniline, 4-aminoantipyrine, and phenol were monitored at 254 nm and, when pyrocatechol–aniline was used as a hydrogen donor, the components of the enzymatic reaction product were monitored at 510 nm [16,17]. Electrochemical analysis Electrochemical measurements were carried out in PBS, 0.2 M, pH 7.0, at room temperature using a Potentiostat/ Galvanostat instrument (Model 263A; EG&G, USA) and a single-compartment voltammetric cell equipped with a platinum rod auxiliary electrode, an Ag/AgCl reference electrode (Metrohm), and a gold working electrode with a disk diameter of 1 mm (Azar Electrode, Uromia, Iran). The preparation of the Au electrode was similar to the procedure previously reported [18–21]. Briefly, the gold electrode was mechanically polished twice with slurries containing alumina powder (particle sizes of 10 and 0.06 lm, respectively) on a polishing microcloth until a mirror finish was observed; then it was sonicated in distilled water for 10 min. Thereafter, the electrode was treated electrochemically in 0.2 M sulfuric acid by potential cycling between 0.2 and 1.5 V (vs Ag/AgCl), at the sweep rate of 100 mV/s, until cyclic voltammogram characteristic of a clean gold electrode was obtained [18–21]. The electrode was then washed with double-distilled water. Results The hydrogen donor couples pyrocatechol–aniline and phenol–aminoantipyrine in the presence of hydrogen peroxide were used as chromogens for HRP assay. Fig. 1 shows a higher sensitivity (DA/min) for pyrocatechol–aniline relative to phenol–aminoantipyrine, which is commonly used for HRP assay [13]. The values depicted in Table 1 also confirm the higher sensitivity and reliability of pyrocatechol–aniline relative to phenol–aminoantipyrine in HRP assay. We also examined the capabilities of other hydrogen donor couples such as pyrocatechol–aminoantipyrine, pyrocatechol–phenol, pyrocatechol–TMPOH, and phenol–aniline as chromogens for HRP assay. However,
40
Spectrophotometric assay for horseradish peroxidase activity / A. Molaei Rad et al. / Anal. Biochem. 362 (2007) 38–43
a
b
Scheme 1. Proposed reaction between aniline and pyrocatechol under (a) electromotive force and (b) HRP biocatalytic activity. All names were obtained by software of ChemDraw Ultra (Cambridge Soft Corp.).
Fig. 1. Sensitivity comparison between two hydrogen donor couples in HRP assay. The absorbance of PBS (0.2 M, pH 7.0, 25 C) containing HRP (1 · 105 mg/ml), H2O2 (1.7 mM), and either (a) aniline (2.5 mM)– pyrocatechol (170 mM) or (b) 4-aminoantipyrine (2.5 mM)–phenol (170 mM) was measured at 510 nm. Inset shows the relationship between absorbance and time.
the color changes produced by these couples were not stable enough to get the reproducible results by UV–Visible spectroscopy (data not shown). To investigate the optimum pH for HRP assay method, DA/min was measured in the pH range of 4.5–8.5 following the procedure mentioned for HRP activity assay. It was observed that DA/min was increased gradually in the pH range from 4.5 to 6.5 and reached a plateau from pH 6.5
to 8.5. Therefore, we chose pH 7.0 PBS throughout this study as the optimum pH which is consistent with that observed by others for soluble peroxidase [22,23]. To examine the repeatability of the method, 20 HRP samples were prepared under the same conditions; then the specific activity of HRP in the samples was calculated using Eq. (1) (Fig. 2). A relative standard deviation (R.S.D.) of 2.9% was obtained for 20 measurements (mean value 931 ± 27 unit/mg), which indicates a satisfactory reproducibility for this method. To determine the detection limit (minimum concentration of HRP that could be detected by this method), DA/ min of enzyme samples was measured while the enzyme concentration was increased gradually. As shown in Fig. 3, this was determined from the cross point of the lines fitted to the linear segments of the rate (DA/min) vs HRP concentration [24]. The value of the detection limit obtained from this experiment was 5.71 · 106 mg/ml. Comparison of this value with that obtained based on the conventional method for HRP assay (Table 1) pointed out the superiority of this method. To consider the characteristics of the dye complex produced by the chromogens, the absorption spectra of the HRP enzymatic reaction in the presence of aniline–pyrocathechol–H2O2 in the wavelength range of 400 to 700 nm were studied (Fig. 4). The absorption spectra at different time intervals revealed that only one absorption peak, with no remarkable shift, was detected in this wavelength range at kmax of 510 nm. It was proposed that a single detectable product was generated during the progress of reaction. To verify this, the reaction mixture was loaded
Spectrophotometric assay for horseradish peroxidase activity / A. Molaei Rad et al. / Anal. Biochem. 362 (2007) 38–43
41
Table 1 Comparison of detection limit and sensitivity obtained from two different hydrogen donor couples for HRP assay Hydrogen donors (AH)
Detection limit for HRPa (mg/ml)
Sensitivity (DA/min)b
Relative standard deviation (R.S.D)c
Pyrocatechol–aniline Phenol–aminoantipyrine
5.71 · 106 9.28 · 106
0.046 ± 0.002 0.010 ± 0.001
3.5% 5.0%
a
Assays were carried out based on the HRP activity assay procedure described under Materials and methods at different HRP concentrations. HRP assays were carried out at HRP concentration of 1 · 105 mg/ml. Other conditions were based on the procedure described under Materials and methods. c R.S.D was calculated from five independent sensitivity measurements (DA/min) under same experimental conditions. b
Fig. 2. Repeatability of HRP assays. The DA/min of enzyme product was measured in aqueous PBS (0.2 M, pH 7.0, 25 C) containing HRP (1 · 105 mg/ml), H2O2 (1.7 mM), and hydrogen donor couple of aniline (2.5 mM)–pyrocatechol (170 mM) at 510 nm.
Fig. 3. Determination of detection limit for HRP. The detection limit was determined from the cross point of the lines fitted to the linear segments of the DA/min (at 510 nm) vs HRP concentration. The experiments were carried out in PBS (0.2 M, pH 7.0, 25 C) containing H2O2 (1.7 mM) and aniline (2.5 mM)–pyrocatechol (170 mM).
into the HPLC column. The resulting chromatogram indicated a single peak at retention time of 2.66 min (Fig. 4, inset). This consequence was in agreement with the data reported previously [25,26].
Fig. 4. Absorption spectra of HRP reaction product. The HRP concentration was 3.28 · 105 mg/ml. The other experimental conditions were the same as Fig. 1a. The spectra from bottom to top were obtained at 30 second intervals. Inset shows the HPLC chromatogram indicating a peak at 2.66 min as the product of enzymatic reaction while the UV–Vis detector was adjusted to 510 nm.
To elucidate how the pyrocatechol–aniline mixture is able to serve as a hydrogen donor mediating couple for HRP, the electrochemical behavior of aniline, pyrocatechol, and the mixture of pyrocatechol–aniline was considered independently using cyclic voltammetry (CV) (Fig. 5). When we used PBS as the background buffer, no redox peak was observed in the potential range between 500 and +500 mV vs. Ag/AgCl (Fig. 5 curve a). By adding 2.5 mM aniline to the PBS almost no electric signal change was detected (Fig. 5 curve b). However, when pyrocatechol (170 mM) was added to the PBS, a cathodic peak was detected at 268 mV vs. Ag/AgCl (Fig. 5 curve c). For the mixture of pyrocatechol (170 mM) and aniline (2.5 mM), a well-defined redox wave with a formal potential of 123 ± 4 mV vs. Ag/AgCl was observed; the cathodic and anodic peak potentials were 214 and 32 mV, respectively, vs. Ag/AgCl (Fig. 5 curve d). The voltammogram of the product of this mixture is a wellcharacterized quasi-reversible redox wave in the solution phase. This product has been used as a redox indicator since its formal potential (0.123 V vs. Ag/AgCl) is close to that of the native HRP (0.220 V vs. Ag/AgCl) in solution [27]. The greatly increased redox peak currents (Fig. 5 curve d) indicated that the mixture of pyrocatechol and aniline electrochemically generates a color component that
42
Spectrophotometric assay for horseradish peroxidase activity / A. Molaei Rad et al. / Anal. Biochem. 362 (2007) 38–43
Fig. 5. Cyclic voltammograms of hydrogen donors. (a) PBS (100 mM, pH 7.0) as the background, (b) 2.5 mM aniline, (c) 170 mM pyrocatechol, and (d) mixture of 170 mM pyrocatechol and 2.5 mM aniline; scan rate 100 mV/s. The electrochemical reactions on the electrode are shown in Scheme 1a.
has an adequate electron transfer ability to serve as a proper mediator for HRP also. As indicated in Fig. 6 and Scheme 1, the absorption spectrum of the dye complex produced in HRP enzymatic reaction in the presence of aniline–pyrocathechol–H2O2 (Fig. 6 curve a) and that produced by the electrochemical process (Fig. 6 curve b) are consistent and both products have the maximum absorption at 510 nm. Fig. 6 also signifies that, although the same concentration of reactants was used, the enzymatic reaction yield was higher than that of
Fig. 7. Lineweaver–Burk plot for HRP. The experiment was carried out in PBS (0.2 M, pH 7.0, 25 C) at a fixed concentration of HRP (1 · 103 mg/ ml), H2O2 (1.7 mM), and aniline (2.5 mM) and different concentrations of pyrocatechol. Inset: Michaelis–Menton plot for HRP.
the electrochemical process. Therefore, with all points taken together, this product could serve as a color indicator for the spectroscopic assay of HRP activity. Fig. 7 represents the Lineweaver–Burk and Michaelis– Menton (Fig. 7 inset) plots for HRP in the presence of pyrocatechol, aniline, and H2O2. Successive addition of pyrocatechol to the solution containing a constant concentration of peroxidase, hydrogen peroxide, and aniline produced a color corresponding to a rather intense visible band at 510 nm. As Fig. 7 inset shows, the reaction rate depends on the initial concentration of pyrocatechol and, at concentrations higher than 150 mM, a steady state is achieved. Based on the intercepts of the Lineweaver–Burk plot, the kinetic parameters of Km and Vmax were determined to be 12.5 mM and 0.0122 mM min1 (12.2 mM min1 mg1), respectively. Discussion
Fig. 6. Comparison between UV–Visible spectra of enzymatic and electrochemical products. (a) Absorption spectrum of the enzymatic reaction product, prepared in PBS (100 mM, pH 7.0) containing HRP (3.28 · 105 mg/ml), H2O2 (1.7 mM), pyrocatechol (170 mM), and aniline (2.5 mM). (b) Absorption spectrum of electrochemical reaction product, prepared in a mixture of pyrocatechol (170 mM) and aniline (2.5 mM) in the presence of H2O2 (1.7 mM). The scan rate was 100 mV/s. For details see text.
There are some similarities between the enzymatic and the electrochemical reactions of pyrocatechol and aniline. Since both reactions produce a pink color component with a kmax of 510 nm (Fig. 6), it seems that both reactions follow the same pathway. It is understandable that the electrochemical reaction of pyrocatechol with aniline begins with the oxidation of pyrocatechol to the quinone species. In the electrochemical pathway, as shown in Scheme 1a, pyrocatechol undergoes a nucleophilic attack by the amine species through a 1,4-Michael addition mechanism [25,26,28]. However, in the case of the enzymatic reaction, a pathway was proposed by which the pyrocatechol substrate is oxidized to free radicals by HRP in the presence of H2O2, as has been shown by ESR [29–31]. Then aniline is coupled with the free radicals of pyrocatechol through a nucleophilic attack by the amine species, followed by the coupling to produce a pink-colored compo-
Spectrophotometric assay for horseradish peroxidase activity / A. Molaei Rad et al. / Anal. Biochem. 362 (2007) 38–43
nent [25,32], as represented in Scheme 1b. Because of the significantly higher sensitivity (Table 1) of pyrocatechol– aniline compared to phenol–aminoantipyrine, pyrocatechol–aniline is suggested to be a better hydrogen donor couple for HRP spectrophotometric assay. In addition, the electrochemical behavior of pyrocatechol–aniline shows that this couple could be used as an adequate electron transfer mediator for HRP. Acknowledgments The authors thank Mrs. Fahime Hatami of the Institute of Biochemistry and Biophysics, University of Tehran for her cooperation in the HPLC experiments of this study. Financial support provided by the Research Council of the University of Tehran and the Iranian National Science Foundation (INSF) is gratefully appreciated. References [1] J.H. Marjon, Van Haandel, M.J.M. Claassens, N.V. Hout, G.M. Boersma, J. Vervoort, M.C.M.I. Rietjens, Differential substrate behavior of phenol and aniline derivatives during conversion by horseradish peroxidase, Biochim. Biophys. Acta 1435 (1999) 22–29. [2] A. Bodtke, W.D. Pfeiffer, N. Ahrensb, P. Langerc, Horseradish peroxidase (HRP) catalyzed oxidative coupling reactions using aqueous hydrogen peroxide: an environmentally benign procedure for the synthesis of azine pigments, Tetrahedron 61 (2005) 1092–10926. [3] R.Z. Harris, S.L. Newmyer, P.R. Ortiz de Montellano, Horseradish peroxidase-catalyzed two-electron oxidations: oxidation of iodide, thioanisoles and phenols at distinct sites, J. Biol. Chem. 268 (1993) 1637–1645. [4] B. Tang, Y. Wang, L. Ma, Simple and rapid catalytic spectrophotometric determination of superoxide anion radical and superoxide dismutase activity in natural medical vegetables using phenol as the substrate for horseradish peroxidase, Anal. Bioanal. Chem. 378 (2004) 523–528. [5] M.A. Azevedo, C.V. Martins, M.F. Duarte Prazeres, V. Vojinovic, M.S. Joaquim Cabral, P.L. Fonseca, Horseradish peroxidase: a valuable tool in biotechnology, Biotechnol. Annu. Rev. 9 (2003) 199–249. [6] E. Agostini, J. Hernandez-Ruiz, B.M. Arnao, R.S. Milrad, A. Horacio Tigier, M. Acosta, A peroxidase isoenzyme secreted by turnip (Brassica napus) hairy-root cultures: inactivation by hydrogen peroxide and application in diagnostic kits, Biotechnol. Appl. Biochem. 35 (2002) 1–7. [7] L. Banci, Structural properties of peroxidases, J. Biotechnol. 53 (1997) 253–258. [8] Q.I. Huang, H.I. Selig, J. Walter, J.R. Weber, Peroxidase-catalyzed oxidative coupling of phenols in the presence of geosorbents: rates of non-extractable product formation, Environ. Sci. Technol. 36 (2002) 596–602. [9] A.C. Maehly, B. Chace, The Assay of Catalases and Peroxidases in Methods of Biochemical Analysis, Interscience Publishers, New York, 1954, I357–358. [10] S. Mukherjeea, T. Weyherm Uller, E. Bothe, K. Wieghardta, P. Chaudhurib, Dinuclear and mononuclear manganese (IV) radical complexes and their catalytic pyrocatecholase activity, Dalton Trans. (2004) 3842–3853. [11] Q. Huang, W.J. Weber, Transformation and removal of bisphenol A from aqueous phase via peroxidase mediated oxidative coupling reactions: efficacy, products, and pathways, Environ. Sci. Technol. 39 (2005) 6029–6036.
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[12] Sigma–Aldrich Co., Sigma–Aldrich Chemie GmbH. Peroxidase from horseradish, (2004) 1570–1571. [13] C.C. Worthington, The Worthington Manual, Worthington Biochemical Co, Lakewood, 1988, 254-257. [14] Mikkivvinodu, M. Padmanabhan, Peroxidase-like catalytic activities of ionic metalloporphyrins supported on functionalised polystyrene surface, Proc. Indian Acad. Sci. (Chem. Sci.) 113 (2001) 1– 9. [15] L. Huang, C. Colas, P.R. Ortiz de Montellano, Oxidation of carboxylic acids by horseradish peroxidase results in prosthetic heme modification and inactivation, J. Am. Chem. Soc. 126 (2004) 2865–12873. [16] K. Hrvoje, N. Koprivanac, B.R. Locke, Decomposition of phenol by hybrid gas/liquid electrical discharge reactors with zeolite catalysts, J. Hazard. Materials B 125 (2005) 190–200. [17] Y. Tao, A. Fishman, E.W. Bentley, K. Thomas, K. Wood, Oxidation of benzene to phenol, pyrocatechol, and 1,2,3-trihydroxybenzene by toluene 4-monooxygenase of Pseudomonas mendocina KR1 and toluene 3-monooxygenase of Ralstonia pickettii PKO1, Appl. Environ. Microbiol. 7 (2004) 3814–3820. [18] Y. Tian, L. Man, T. Okajima, T. Ohsaka, Superoxide dismutasebased third-generation biosensor for superoxide anion, Anal. Chem. 74 (2002) 2428–2434. [19] Y. Tian, M. Shioda, S. Kasahara, T. Okajima, L. Mao, T. Hisabori, T. Ohsaka, A facilitated electron transfer of copper–zinc superoxide dismutase (SOD) based on a cysteine-bridged SOD electrode, Biochim. Biophys. Acta 1569 (2002) 151–158. [20] Y. Tian, T. Ariga, N. Takashima, T. Okajima, Self-assembled monolayer suitable for electron-transfer promotion of copper, zinc-superoxide dismutase, Electrochem. Commun. 6 (2004) 609– 614. [21] J. Hong, H. Ghourchian, A.A. Moosavi–Movahedi, Direct electron transfer of redox proteins on a nafion-cysteine modified gold electrode, Electrochem. Commun. 8 (2006) 1572–1576. [22] A.C. Maehly, Plant peroxidases: Methods in Enzymology, Academic Press, New York, 1995, 807. [23] H. Yao, N. Li, S. Xu, J.Z. Xu, J.J. Zhu, H.Y. Chen, Electrochemical study of a new methylene blue/silicon oxide nanocomposition mediator and its application for stable biosensor of hydrogen peroxide, Biosens. Bioelectron. 21 (2005) 372–377. [24] R.P. Buck, E. Linder, Recommendations for nomenclature of ion selective electrodes, Pure Appl. Chem. 66 (1994) 2527–2536. [25] E.H. Seymour, S. Nathan Lawrence, L. Emma Beckett, J. Davis, R.G. Compton, Electrochemical detection of aniline: an electrochemically initiated reaction pathway, Talanta 57 (2002) 233–242. [26] S. Uchiyama, Y. Hasebe, J. Nishimoto, H. Hamana, Y. Maeda, Y. Yoshida, Formation of aniline-bonded catechols in aqueous solution and their electrochemical behavior at a carbon felt electrode, Electroanalysis 10 (1998) 647–650. [27] H. A Harbury, Oxidation–reduction potentials of horseradish peroxidase, J. Biol. Chem. 225 (1957) 1009–1024. [28] A. De Riso, M. Gullotti, L. Casella, E. Monzani, Selectivity in the peroxidase catalyzed oxidation of phenolic sulfides, J. Mol. Catalysis; A: Chemical 204 (2003) 391–400. [29] M. Wagner, J.A. Nicell, Detoxification of phenolic solutions with horseradish peroxidase and hydrogen peroxide, Water Res. 36 (2002) 4041–4052. [30] L. Huang, G. Wojciechowski, P.R. Ortiz de Montellano, Prosthetic heme modification during halide ion oxidation. Demonstration of chloride oxidation by horseradish peroxidase, J. Am. Chem. Soc. 127 (2005) 5345–5353. [31] J. Dec, K. Haider, J. Marc Bollag, Release of substituents from phenolic compounds during oxidative coupling reactions, Chemosphere 52 (2003) 549–556. [32] W. Liu, J. Kumar, S. Tripathy, K.J. Senecal, L. Samuelson, Enzymatically synthesized conducting polyaniline, J. Am. Chem. Soc. 121 (1999) 71–78.
Spectrophotometric assay for horseradish peroxidase activity based on pyrocatechol–aniline coupling hydrogen donor A. Molaei Rad a, H. Ghourchian a
a,*
, A.A. Moosavi-Movahedi a, J. Hong a, K. Nazari
b
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran b Research Institute of Petroleum Industry, Tehran, Iran Received 27 August 2006 Available online 20 December 2006
Abstract The hydrogen donor couples pyrocatechol–aniline and phenol–aminoantipyrine in the presence of hydrogen peroxide were compared as chromogens for horseradish peroxidase (HRP) assay. UV–Visible spectroscopy and high-performance liquid chromatography analysis indicated that during the HRP biocatalytic process, pyrocatechol–aniline was converted to a pink-colored reagent with a kmax of 510 nm, which was used in the assay of HRP activity. Electrochemical studies revealed adequate electron transfer ability for this color reagent to serve as a proper mediator for HRP also. Using pyrocatechol–aniline a higher sensitivity and lower detection limit was obtained relative to those of the phenol–aminoantipyrine couple, which is commonly used for HRP assay. A relative standard deviation of 2.9% was obtained for 20 HRP activity measurements, indicating a satisfactory reproducibility for this method. In addition, kinetic parameters of Km (12.5 mM) and Vmax (12.2 mM min1 mg1) were calculated for pyrocatechol–aniline. Regarding the superiority of pyrocatechol–aniline, this couple is suggested to be a better hydrogen donor for the HRP spectrophotometric assay. 2006 Elsevier Inc. All rights reserved. Keywords: Horseradish peroxidase; Hydrogen donor; Pyrocatechol; Aniline; Enzyme assay
Horseradish peroxidase (EC 1.11.1.7; donor-H2O2 oxidoreductase) (HRP)1 is one of the heme peroxidases, which catalyze a variety of oxidative transformations of organic and inorganic substrates by hydrogen peroxide or alkyl peroxides [1–7]. Furthermore, most phenolic compounds are subject to oxidative coupling and the reaction can be catalyzed by certain naturally occurring extracellular enzymes and peroxidases [8]. The activity of these peroxidases has traditionally been expressed in units based upon the rate of oxidation of pyrogallol introduced by Willstalter and Stoll in 1917. The simplicity, rapidity, facile and *
Corresponding author. Fax: +98 21 66404680. E-mail address: [email protected] (H. Ghourchian). 1 Abbreviations used: HRP, horseradish peroxidase; HPLC, high-performance liquid chromatography; ESR, electron spin resonance; LC, liquid chromatography; ABTS, 2,2-azino-bis(3-ethylbenzothiazolin-6-sulfonate); TMPOH, 2,2,6,6-tetramethyl piperidone-oxime hydrochloride; PBS, phosphate buffer solution; CV, cyclic voltammetry; R.S.D., relative standard deviation. 0003-2697/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.11.035
inexpensive properties of spectrophotometry have made it a popular method to determine activity of HRP [9], therefore more detailed studies by using this technique have been presented since 1954 [10]. A wide variety of hydrogen donors (e.g., caffeic acid and 2,2-azino-bis(3-ethylbenzothiazolin-6-sulfonate)) have been utilized in peroxidase assay systems including methods using potentially carcinogenic and mutagenic materials [11,12]. In addition, an improved spectrophotometric assay has been adopted using 4-aminoantipyrine and phenol as hydrogen donors, although phenol itself is carcinogenic and must be handled with extra precautions. In this method, the reaction rate is determined by measuring the increase in absorbance caused by the formation of a dye complex resulting from the decomposition of hydrogen peroxide [13]. In the present study, we have proposed the pyrocatechol– aniline couple as hydrogen donor for horseradish peroxidase assay and developed an improved spectrophotometric method for the determination of HRP activity.
Spectrophotometric assay for horseradish peroxidase activity / A. Molaei Rad et al. / Anal. Biochem. 362 (2007) 38–43
Materials and methods Reagents Horseradish peroxidase types II with a purity index (RZ) of 2.0, hydrogen peroxide, and finer alumina powder were obtained from Sigma. Phenol, 4-aminoantipyrine, sodium phosphate, pyrocatechol (1,2-dihydroxybezene), 2,2,6, 6-tetramethyl piperidone-oxime hydrochloride (TMPOH), aniline, and acetonitrile (99.8%) were obtained from Merck. All solutions were prepared in double-distilled deionized water (Barnstead Nanopure D4742, resistance 18.3 MX). HRP activity assay The activity of the enzyme was determined colorimetrically using a UV–Vis spectrophotometer (Cary 100 Bio, Varian, Australia), equipped with a temperature controller. A mixture of pyrocatechol (170 mM) and aniline (2.5 mM) was prepared in phosphate buffer solution (PBS) 0.2 M, pH 7.0. To each blank and sample cuvettes, 450 ll of the above mixture solution and 500 ll of hydrogen peroxide (1.7 mM) were pipetted. The spectrophotometer was adjusted to 510 nm. To achieve a temperature equilibration both mixtures in cuvettes were incubated in spectrophotometer at 25 C for 3–4 min. Then, 50 ll of diluted HRP and 50 ll PBS were added to the sample and blank cuvettes, respectively. Increase in absorbance was recorded for 4–5 min. The concentration of HRP should be so diluted that a certain reaction rate (usually, DA/min = 0.02–0.04) could be obtained. The reaction rate was determined by measuring the increase in absorbance resulting from the decomposition of hydrogen peroxide and formation of the colored product of 4-(phenylamino)benzene-1,2-diol (Scheme 1b). Prior to each assay, the concentration of hydrogen peroxide was determined based on its absorbance at 240 nm and extinction coefficient (e240 = 43.6 cm1 M1) [14,15]. The activity of HRP was calculated based on Eq. (1), units=mg ¼
DA= min ; e mgðenzymeÞ=mlðreaction mixtureÞ
ð1Þ
where e (extinction coefficient) for 4-(phenylamino)benzene-1,2-diol is 5 mM1 cm1. To evaluate the performance of this method, the obtained results were compared with those acquired from the conventional method depicted in the Worthington manual [13]. To estimate the capability of other hydrogen donor couples in HRP assay, the same procedure was used except that the spectrophotometer was adjusted to 360 nm for pyrocatechol–aminoantipyrine and to 310 nm for pyrocatechol–phenol, pyrocatechol–TMPOH, and phenol– aniline. HPLC analysis High-performance liquid chromatography separation was performed on a Chromatopac Shimadzu C-R4A, Sys-
39
tem controller SCL-6A, equipped with a UV–Vis spectrophotometer detector Model SPD-6AV, liquid chromatograph LC-6A, and a LiChrospher 100 column, RP-18 (5-lm particle size)-LiChroCART 250-4. A solution of PBS (0.2 M, pH 7) with total volume of 1 ml containing H2O2(1.7 mM), pyrocatechol (170 mM) and aniline (2.5 mM) was prepared at 25 C. To avoid column contamination by the enzyme, the HRP solution was kept in a dialysis bag, cutoff 10 kDa, while it was exposed to the reaction mixture. A pink-colored component was formed gradually. Then, 20 ll of the mixture was directly injected into the column. A mixture of acetonitrile–water with a flow rate of 1.0 ml/min was used as the mobile phase with a ratio of 10/90 (v/v) at starting point. The ratio was gradually changed to 90/10 (v/v) within a period of 60 min. Chromatograms for the determination of pyrocatechol, aniline, 4-aminoantipyrine, and phenol were monitored at 254 nm and, when pyrocatechol–aniline was used as a hydrogen donor, the components of the enzymatic reaction product were monitored at 510 nm [16,17]. Electrochemical analysis Electrochemical measurements were carried out in PBS, 0.2 M, pH 7.0, at room temperature using a Potentiostat/ Galvanostat instrument (Model 263A; EG&G, USA) and a single-compartment voltammetric cell equipped with a platinum rod auxiliary electrode, an Ag/AgCl reference electrode (Metrohm), and a gold working electrode with a disk diameter of 1 mm (Azar Electrode, Uromia, Iran). The preparation of the Au electrode was similar to the procedure previously reported [18–21]. Briefly, the gold electrode was mechanically polished twice with slurries containing alumina powder (particle sizes of 10 and 0.06 lm, respectively) on a polishing microcloth until a mirror finish was observed; then it was sonicated in distilled water for 10 min. Thereafter, the electrode was treated electrochemically in 0.2 M sulfuric acid by potential cycling between 0.2 and 1.5 V (vs Ag/AgCl), at the sweep rate of 100 mV/s, until cyclic voltammogram characteristic of a clean gold electrode was obtained [18–21]. The electrode was then washed with double-distilled water. Results The hydrogen donor couples pyrocatechol–aniline and phenol–aminoantipyrine in the presence of hydrogen peroxide were used as chromogens for HRP assay. Fig. 1 shows a higher sensitivity (DA/min) for pyrocatechol–aniline relative to phenol–aminoantipyrine, which is commonly used for HRP assay [13]. The values depicted in Table 1 also confirm the higher sensitivity and reliability of pyrocatechol–aniline relative to phenol–aminoantipyrine in HRP assay. We also examined the capabilities of other hydrogen donor couples such as pyrocatechol–aminoantipyrine, pyrocatechol–phenol, pyrocatechol–TMPOH, and phenol–aniline as chromogens for HRP assay. However,
40
Spectrophotometric assay for horseradish peroxidase activity / A. Molaei Rad et al. / Anal. Biochem. 362 (2007) 38–43
a
b
Scheme 1. Proposed reaction between aniline and pyrocatechol under (a) electromotive force and (b) HRP biocatalytic activity. All names were obtained by software of ChemDraw Ultra (Cambridge Soft Corp.).
Fig. 1. Sensitivity comparison between two hydrogen donor couples in HRP assay. The absorbance of PBS (0.2 M, pH 7.0, 25 C) containing HRP (1 · 105 mg/ml), H2O2 (1.7 mM), and either (a) aniline (2.5 mM)– pyrocatechol (170 mM) or (b) 4-aminoantipyrine (2.5 mM)–phenol (170 mM) was measured at 510 nm. Inset shows the relationship between absorbance and time.
the color changes produced by these couples were not stable enough to get the reproducible results by UV–Visible spectroscopy (data not shown). To investigate the optimum pH for HRP assay method, DA/min was measured in the pH range of 4.5–8.5 following the procedure mentioned for HRP activity assay. It was observed that DA/min was increased gradually in the pH range from 4.5 to 6.5 and reached a plateau from pH 6.5
to 8.5. Therefore, we chose pH 7.0 PBS throughout this study as the optimum pH which is consistent with that observed by others for soluble peroxidase [22,23]. To examine the repeatability of the method, 20 HRP samples were prepared under the same conditions; then the specific activity of HRP in the samples was calculated using Eq. (1) (Fig. 2). A relative standard deviation (R.S.D.) of 2.9% was obtained for 20 measurements (mean value 931 ± 27 unit/mg), which indicates a satisfactory reproducibility for this method. To determine the detection limit (minimum concentration of HRP that could be detected by this method), DA/ min of enzyme samples was measured while the enzyme concentration was increased gradually. As shown in Fig. 3, this was determined from the cross point of the lines fitted to the linear segments of the rate (DA/min) vs HRP concentration [24]. The value of the detection limit obtained from this experiment was 5.71 · 106 mg/ml. Comparison of this value with that obtained based on the conventional method for HRP assay (Table 1) pointed out the superiority of this method. To consider the characteristics of the dye complex produced by the chromogens, the absorption spectra of the HRP enzymatic reaction in the presence of aniline–pyrocathechol–H2O2 in the wavelength range of 400 to 700 nm were studied (Fig. 4). The absorption spectra at different time intervals revealed that only one absorption peak, with no remarkable shift, was detected in this wavelength range at kmax of 510 nm. It was proposed that a single detectable product was generated during the progress of reaction. To verify this, the reaction mixture was loaded
Spectrophotometric assay for horseradish peroxidase activity / A. Molaei Rad et al. / Anal. Biochem. 362 (2007) 38–43
41
Table 1 Comparison of detection limit and sensitivity obtained from two different hydrogen donor couples for HRP assay Hydrogen donors (AH)
Detection limit for HRPa (mg/ml)
Sensitivity (DA/min)b
Relative standard deviation (R.S.D)c
Pyrocatechol–aniline Phenol–aminoantipyrine
5.71 · 106 9.28 · 106
0.046 ± 0.002 0.010 ± 0.001
3.5% 5.0%
a
Assays were carried out based on the HRP activity assay procedure described under Materials and methods at different HRP concentrations. HRP assays were carried out at HRP concentration of 1 · 105 mg/ml. Other conditions were based on the procedure described under Materials and methods. c R.S.D was calculated from five independent sensitivity measurements (DA/min) under same experimental conditions. b
Fig. 2. Repeatability of HRP assays. The DA/min of enzyme product was measured in aqueous PBS (0.2 M, pH 7.0, 25 C) containing HRP (1 · 105 mg/ml), H2O2 (1.7 mM), and hydrogen donor couple of aniline (2.5 mM)–pyrocatechol (170 mM) at 510 nm.
Fig. 3. Determination of detection limit for HRP. The detection limit was determined from the cross point of the lines fitted to the linear segments of the DA/min (at 510 nm) vs HRP concentration. The experiments were carried out in PBS (0.2 M, pH 7.0, 25 C) containing H2O2 (1.7 mM) and aniline (2.5 mM)–pyrocatechol (170 mM).
into the HPLC column. The resulting chromatogram indicated a single peak at retention time of 2.66 min (Fig. 4, inset). This consequence was in agreement with the data reported previously [25,26].
Fig. 4. Absorption spectra of HRP reaction product. The HRP concentration was 3.28 · 105 mg/ml. The other experimental conditions were the same as Fig. 1a. The spectra from bottom to top were obtained at 30 second intervals. Inset shows the HPLC chromatogram indicating a peak at 2.66 min as the product of enzymatic reaction while the UV–Vis detector was adjusted to 510 nm.
To elucidate how the pyrocatechol–aniline mixture is able to serve as a hydrogen donor mediating couple for HRP, the electrochemical behavior of aniline, pyrocatechol, and the mixture of pyrocatechol–aniline was considered independently using cyclic voltammetry (CV) (Fig. 5). When we used PBS as the background buffer, no redox peak was observed in the potential range between 500 and +500 mV vs. Ag/AgCl (Fig. 5 curve a). By adding 2.5 mM aniline to the PBS almost no electric signal change was detected (Fig. 5 curve b). However, when pyrocatechol (170 mM) was added to the PBS, a cathodic peak was detected at 268 mV vs. Ag/AgCl (Fig. 5 curve c). For the mixture of pyrocatechol (170 mM) and aniline (2.5 mM), a well-defined redox wave with a formal potential of 123 ± 4 mV vs. Ag/AgCl was observed; the cathodic and anodic peak potentials were 214 and 32 mV, respectively, vs. Ag/AgCl (Fig. 5 curve d). The voltammogram of the product of this mixture is a wellcharacterized quasi-reversible redox wave in the solution phase. This product has been used as a redox indicator since its formal potential (0.123 V vs. Ag/AgCl) is close to that of the native HRP (0.220 V vs. Ag/AgCl) in solution [27]. The greatly increased redox peak currents (Fig. 5 curve d) indicated that the mixture of pyrocatechol and aniline electrochemically generates a color component that
42
Spectrophotometric assay for horseradish peroxidase activity / A. Molaei Rad et al. / Anal. Biochem. 362 (2007) 38–43
Fig. 5. Cyclic voltammograms of hydrogen donors. (a) PBS (100 mM, pH 7.0) as the background, (b) 2.5 mM aniline, (c) 170 mM pyrocatechol, and (d) mixture of 170 mM pyrocatechol and 2.5 mM aniline; scan rate 100 mV/s. The electrochemical reactions on the electrode are shown in Scheme 1a.
has an adequate electron transfer ability to serve as a proper mediator for HRP also. As indicated in Fig. 6 and Scheme 1, the absorption spectrum of the dye complex produced in HRP enzymatic reaction in the presence of aniline–pyrocathechol–H2O2 (Fig. 6 curve a) and that produced by the electrochemical process (Fig. 6 curve b) are consistent and both products have the maximum absorption at 510 nm. Fig. 6 also signifies that, although the same concentration of reactants was used, the enzymatic reaction yield was higher than that of
Fig. 7. Lineweaver–Burk plot for HRP. The experiment was carried out in PBS (0.2 M, pH 7.0, 25 C) at a fixed concentration of HRP (1 · 103 mg/ ml), H2O2 (1.7 mM), and aniline (2.5 mM) and different concentrations of pyrocatechol. Inset: Michaelis–Menton plot for HRP.
the electrochemical process. Therefore, with all points taken together, this product could serve as a color indicator for the spectroscopic assay of HRP activity. Fig. 7 represents the Lineweaver–Burk and Michaelis– Menton (Fig. 7 inset) plots for HRP in the presence of pyrocatechol, aniline, and H2O2. Successive addition of pyrocatechol to the solution containing a constant concentration of peroxidase, hydrogen peroxide, and aniline produced a color corresponding to a rather intense visible band at 510 nm. As Fig. 7 inset shows, the reaction rate depends on the initial concentration of pyrocatechol and, at concentrations higher than 150 mM, a steady state is achieved. Based on the intercepts of the Lineweaver–Burk plot, the kinetic parameters of Km and Vmax were determined to be 12.5 mM and 0.0122 mM min1 (12.2 mM min1 mg1), respectively. Discussion
Fig. 6. Comparison between UV–Visible spectra of enzymatic and electrochemical products. (a) Absorption spectrum of the enzymatic reaction product, prepared in PBS (100 mM, pH 7.0) containing HRP (3.28 · 105 mg/ml), H2O2 (1.7 mM), pyrocatechol (170 mM), and aniline (2.5 mM). (b) Absorption spectrum of electrochemical reaction product, prepared in a mixture of pyrocatechol (170 mM) and aniline (2.5 mM) in the presence of H2O2 (1.7 mM). The scan rate was 100 mV/s. For details see text.
There are some similarities between the enzymatic and the electrochemical reactions of pyrocatechol and aniline. Since both reactions produce a pink color component with a kmax of 510 nm (Fig. 6), it seems that both reactions follow the same pathway. It is understandable that the electrochemical reaction of pyrocatechol with aniline begins with the oxidation of pyrocatechol to the quinone species. In the electrochemical pathway, as shown in Scheme 1a, pyrocatechol undergoes a nucleophilic attack by the amine species through a 1,4-Michael addition mechanism [25,26,28]. However, in the case of the enzymatic reaction, a pathway was proposed by which the pyrocatechol substrate is oxidized to free radicals by HRP in the presence of H2O2, as has been shown by ESR [29–31]. Then aniline is coupled with the free radicals of pyrocatechol through a nucleophilic attack by the amine species, followed by the coupling to produce a pink-colored compo-
Spectrophotometric assay for horseradish peroxidase activity / A. Molaei Rad et al. / Anal. Biochem. 362 (2007) 38–43
nent [25,32], as represented in Scheme 1b. Because of the significantly higher sensitivity (Table 1) of pyrocatechol– aniline compared to phenol–aminoantipyrine, pyrocatechol–aniline is suggested to be a better hydrogen donor couple for HRP spectrophotometric assay. In addition, the electrochemical behavior of pyrocatechol–aniline shows that this couple could be used as an adequate electron transfer mediator for HRP. Acknowledgments The authors thank Mrs. Fahime Hatami of the Institute of Biochemistry and Biophysics, University of Tehran for her cooperation in the HPLC experiments of this study. Financial support provided by the Research Council of the University of Tehran and the Iranian National Science Foundation (INSF) is gratefully appreciated. References [1] J.H. Marjon, Van Haandel, M.J.M. Claassens, N.V. Hout, G.M. Boersma, J. Vervoort, M.C.M.I. Rietjens, Differential substrate behavior of phenol and aniline derivatives during conversion by horseradish peroxidase, Biochim. Biophys. Acta 1435 (1999) 22–29. [2] A. Bodtke, W.D. Pfeiffer, N. Ahrensb, P. Langerc, Horseradish peroxidase (HRP) catalyzed oxidative coupling reactions using aqueous hydrogen peroxide: an environmentally benign procedure for the synthesis of azine pigments, Tetrahedron 61 (2005) 1092–10926. [3] R.Z. Harris, S.L. Newmyer, P.R. Ortiz de Montellano, Horseradish peroxidase-catalyzed two-electron oxidations: oxidation of iodide, thioanisoles and phenols at distinct sites, J. Biol. Chem. 268 (1993) 1637–1645. [4] B. Tang, Y. Wang, L. Ma, Simple and rapid catalytic spectrophotometric determination of superoxide anion radical and superoxide dismutase activity in natural medical vegetables using phenol as the substrate for horseradish peroxidase, Anal. Bioanal. Chem. 378 (2004) 523–528. [5] M.A. Azevedo, C.V. Martins, M.F. Duarte Prazeres, V. Vojinovic, M.S. Joaquim Cabral, P.L. Fonseca, Horseradish peroxidase: a valuable tool in biotechnology, Biotechnol. Annu. Rev. 9 (2003) 199–249. [6] E. Agostini, J. Hernandez-Ruiz, B.M. Arnao, R.S. Milrad, A. Horacio Tigier, M. Acosta, A peroxidase isoenzyme secreted by turnip (Brassica napus) hairy-root cultures: inactivation by hydrogen peroxide and application in diagnostic kits, Biotechnol. Appl. Biochem. 35 (2002) 1–7. [7] L. Banci, Structural properties of peroxidases, J. Biotechnol. 53 (1997) 253–258. [8] Q.I. Huang, H.I. Selig, J. Walter, J.R. Weber, Peroxidase-catalyzed oxidative coupling of phenols in the presence of geosorbents: rates of non-extractable product formation, Environ. Sci. Technol. 36 (2002) 596–602. [9] A.C. Maehly, B. Chace, The Assay of Catalases and Peroxidases in Methods of Biochemical Analysis, Interscience Publishers, New York, 1954, I357–358. [10] S. Mukherjeea, T. Weyherm Uller, E. Bothe, K. Wieghardta, P. Chaudhurib, Dinuclear and mononuclear manganese (IV) radical complexes and their catalytic pyrocatecholase activity, Dalton Trans. (2004) 3842–3853. [11] Q. Huang, W.J. Weber, Transformation and removal of bisphenol A from aqueous phase via peroxidase mediated oxidative coupling reactions: efficacy, products, and pathways, Environ. Sci. Technol. 39 (2005) 6029–6036.
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