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  1. 1. Biochimie 86 (2004) 519–522 www.elsevier.com/locate/biochi Tannic acid interferes with the commonly used laccase-detection assay based on ABTS as the substrate M.C. Terrón, M. López-Fernández, J.M. Carbajo 1, H. Junca 2, A. Téllez 3, S. Yagüe, A. Arana-Cuenca 3, T. González 4, A.E. González * Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040, Madrid, Spain Received 10 May 2004; accepted 16 July 2004 Available online 20 August 2004 Abstract Laccase enzymatic activity in biological samples is usually detected spectrophotometrically through its capacity to oxidize several specific aromatic compounds. One of the most commonly used substrates is the compound 2-2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), which becomes green-blue coloured when it is oxidized by laccase. In this work we study the interference of tannic acid with the spectrophotometric assay to detect laccase by using ABTS as the substrate. Our data show that under the normal reaction conditions of this assay, but in the absence of any catalyst, tannic acid is able to carry out the chemical reduction of the oxidized specie of ABTS, thus decreasing the overall detectable laccase-activity values observed when this enzyme is present in the reaction mixture. Therefore, our results represent an important warning concerning a commonly used method for measuring, detecting or screening laccases in biological samples that may content tannic acid or structural-related molecules. © 2004 Elsevier SAS. All rights reserved. Keywords: Laccase; Laccase-detection; ABTS; Tannic acid 1. Introduction well as low molecular weight toxic phenols [3], industrial dyes [4], chlorophenols [5], together with anthracene, phe- Laccases (benzenediol: oxygen oxidoreductase, EC nanthrene and other polycyclic aromatic hydrocarbons are multi-copper phenoloxidases detected in many (PAHs) [6,7]. The rather broad substrate specificity of fungal plants and secreted by numerous fungi. They catalyse the laccases has generated an increased interest in a variety of oxidation of a number of quite different aromatic substances different biotechnological applications for this methalloen- (diphenols, methoxy-substituted monophenols, aromatic zyme. Currently laccases are used in pulp delignification, amines) using oxygen as the final electron acceptor [1,2]. textile dye bleaching, bioremediation and effluent detoxifica- Laccases from some basidiomycetous fungi have been tion, as well as being used in detergents and in biosensors, shown to be effective in oxidizing a number of pollutants as among other applications [8,9]. Laccase activity is commonly determined spectrophoto- metrically based on the capacity of this enzyme to oxidize– colorize specific aromatic compounds such as guaiacol, sy- Abbreviations: ABTS, 2-2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid); TA, Tannic acid; 2,6-DMP, 2,6-dimethoxyphenol. ringaldazine [10], or 2,6-dimethoxyphenol (2,6-DMP) [11]. * Corresponding author. Tel.: +34-91-8373112 Ext. 4413; One of the most commonly used substrates is the electron- fax: +34-91-5360432. rich non-phenolic compound 2-2′-azinobis(3-ethylbenzthia- E-mail address: aldo@cib.csic.es (A.E. González). zoline-6-sulfonic acid) (ABTS) [12], because, in contrast to 1 INIA, Carretera de la Coruña Km 7.5, 28040 Madrid, Spain the phenolic substrates forming quinones, the oxidation po- 2 GBF-National Research Centre for Biotechnology, D-38124 Braun- tential of ABTS is not pH-dependent within the range 2–11 schweig, Germany 3 Universidad Politécnica de Pachuca, Zenpoala. C.P. 43830. Estado de [13] and proceeds in one step. Hidalgo, México Oxidation of ABTS by laccase results in the production of 4 Instituto Cubano de Derivados de la Caña de Azúcar, Havana, Cuba a green–blue coloured radical cation (ABTS+•) measurable 0300-9084/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2004.07.013
  2. 2. 520 M.C. Terrón et al. / Biochimie 86 (2004) 519–522 at 436 nm (eo= 29300 M–1 cm–1). The reaction mixture as described above but without the addition of TA. Laccase usually consists of acetate buffer at around pH 5, ABTS as activity was measured immediately after the addition of TA the substrate (10 mM final concentration), and the fungal solutions to enzymatic crudes using both, ABTS and 2,6- extracellular medium containing the laccase activity to be DMP as substrates. measured. In this work, we demonstrate that under these reaction 2.4. Laccase activity determination conditions but in the absence of any enzyme, tannic acid (TA) is able to carry out the chemical reduction of ABTS+•. This Laccase activity was measured, using 2,6-DMP [11] or results in an underestimation of laccase activity values deter- ABTS [12] as enzyme substrates. One unit of laccase activity mined by this method in biological samples containing TA or is defined as the formation of 1 µmol of product per min. All related aromatic compounds. assays were performed in duplicate using a Shimadzu UV- 1603 spectrophotometer. 2. Materials and methods 2.5. ABTS Spectra 2.1. Chemicals The absorption spectra of an ABTS (10 mM) solution dissolved in water was carried out in a Jasco V-530 spectro- TA (Tannic acid powder pure, USP, empirical formula photometer from 400 to 500 nm. Different ABTS spectra C76H52O46), was obtained from Merck (Darmstadt, Ger- were carried out in the absence and in the presence of TA many). ABTS was purchased from Boehringer-Mannheim (0.25, 0.50, and 1 µM final concentrations) or sodium ascor- (Germany) and 2,6-DMP from Fluka (Germany). All other bate (25 µM). chemicals were reagent grade obtained from Merck or Sigma-Aldrich. 3. Results and discussion 2.2. Organism and maintenance The interference in the laccase-detection assay using Basidiomycetes Coriolopsis gallica (A-241) and Tram- ABTS as the substrate (evidenced by the decrease of absor- etes sp. I-62 (B-24), were obtained from the IJFM (Instituto bance at 436 nm in the presence of different industrial efflu- Jaime Ferrán de Microbiología) collection. The fungal cul- ents), is a frequent observation in our laboratory (data not tures were maintained on malt agar slants (2% malt extract, published). Given that all these wastewaters come from in- 2% Bacto Agar), grown for 10 days at 28 °C, and stored at dustries that use plants as raw material, we speculated that 4 °C. the common substances which may be responsible for this interference could be polyphenolic based compounds; which 2.3. Culture conditions in addition are molecules which could be easily oxidized by the enzyme. Thus, we determined the total phenol and tannin C. gallica was grown on agar plates with modified Cza- content in some of these effluents, which indicated that tan- peck medium [14] for seven days at 28 °C. Ten plugs (1 cm2) nins represent a significant percentage of their phenolic com- were cut and inoculated under sterile conditions into 500 ml position [16]. For this reason, TA (the most abundant culture flasks containing 300 ml of Kirk growth medium with polyphenolic molecule in plants after lignin) was selected as 0.4 mM veratryl alcohol [15]. After incubation for 48 h at a model compound in our study. 28 °C in an orbital shaker (200 rpm), an inoculum of 1:10 Experiments were performed initially to elucidate (v/v) was transferred into 250 ml Erlenmeyer flasks contain- whether the observed interference with laccase activity ing 100 ml of the same medium. Samples were incubated at might be due to a direct interference with the laccase enzyme 28 °C and 125 rpm for 7 days. Afterwards, a filter-sterilized itself, or to a chemical interference with ABTS. With this in (0.22 µm) water-solution of 50 mM TA was added to the mind, we monitored laccase activity in C. gallica over an culture medium to reach a final concentration of 200 µM, and 8-day period; grown in either the presence or absence of the cultures were incubated under the same conditions for 200 µM of TA. Laccase activity was measured using ABTS eight more days. During this time laccase activity was mea- or 2,6-DMP (Fig. 1). Significant differences were observed sured daily in the extracellular fluids using ABTS and 2,6- with much higher laccase activity values being detected in DMP as substrates. Controls without TA were also run and the presence of TA using 2,6-DMP as substrate as opposed to monitored daily. ABTS. For example, laccase activity determined with 2,6- The assays to determine laccase activity in the presence of DMP was 3.2-fold higher than in the control without TA, in different concentrations of TA (0, 0.1, 1, 5, 9, 20, 50, 100 and day 6 samples. On the other hand, laccase activity deter- 200 µM, final concentrations) were performed by mixing mined with ABTS was 9-fold lower than that observed on the them with extracellular fluids from 12-days-old cultures of same day in the control. These results suggested the possible Trametes sp. I-62, grown under the same culture conditions interference of tannic acid with the ABTS molecule. This is
  3. 3. M.C. Terrón et al. / Biochimie 86 (2004) 519–522 521 Fig. 1. Laccase activity determined as oxidation of (n) 2,6-DMP or (•) ABTS in the extracellular fluid of Coriolopsis gallica. Each point repre- sents: [100 × (laccase activity measured in the presence of 200 µM of TA/laccase activity in the control without TA, measured the same day)]. supported by a previous observation by Carbajo and cowork- ers [17] where increased laccase activity was observed when TA (100 µM) was added to C. gallica cultures, suggesting that TA interferes with ABTS molecule rather than inhibit laccase production in this fungus. These results prompted us to perform additional analysis to verify this interference. The absorption spectra of a solu- Fig. 2. (A) Optical absorption spectra of ABTS in the absence (a) and in the tion of ABTS both in the presence and absence of different presence of different concentrations of TA (0.25 (b), 0.50 (c), and 1 µM (d)) concentrations of TA were then assessed (Fig. 2A). The or sodium ascorbate (25 µM) (e). (B) Laccase activity determined using (n) absorbance values of ABTS were markedly lower especially 2,6-DMP or (•) ABTS in the presence of different concentrations of TA. For the experiment, extracellular fluids containing high laccase activity from in the 400–460 nm range, at increased concentrations of TA. 12-days old cultures of Trametes sp. I-62 were used. Moreover at TA concentrations of 0.5 µM and higher, the characteristic shoulder around 420 nm was absent. The spec- the absence of TA, even in the presence of the highest TA trum of ABTS in the presence of 25 µM ascorbate, a well- concentration assayed (200 µM). known chemical ABTS reducer [12], was also performed; Recently, other organic compounds have also been re- and were shown to be very similar to those of ABTS in the ported to be capable of reducing ABTS. Johannes and Ma- presence of 0.5 and 1 µM TA (Fig. 2A). This strongly sug- jcherczyk [18] have demonstrated that several sulfhydryl gests that TA can chemically reduce the ABTS molecule. It is organic compounds, described as laccase inhibitors, are not well established that at ABTS concentrations greater than true inhibitors but rather are substances which are able to 1 mM, solutions appear green-blue but become colourless chemically reduce the coloured radical cation ABTS+• to when reducing agents such as ascorbate or cysteamine are ABTS, resulting in the decolourising of the solution. The added, even at relatively low concentrations [12]. A similar reduction of ABTS by some physiological organic acids and change in colour was also observed here when TA was added a phenolic lignin-related compound has also been described to ABTS. [19]. In addition we performed assays on a native laccase en- The results presented here indicate that TA is also capable zyme crude from an 12-day-old culture of Trametes sp. I-62, of chemically reducing ABTS+• in reaction conditions com- in order to verify whether TA–ABTS interference also af- monly used to detect laccase activity, leading to the possibil- fected enzymatic activity measurements. Laccase activity ity that much lower laccase activity values will be obtained was measured both in the presence and absence of different when this compound is present in the reaction mixture. This concentrations of TA and using either ABTS or 2,6-DMP as interference could probably be extended to include other substrates (Fig. 2B). The results obtained indicated a dra- easily oxidised structural-related phenolic compounds; and matic decrease in ABTS mediated laccase activity following may explain at least in part, the decrease in absorbance addition of 5 µM TA, reaching minimum values (less that 5% observed in the presence of some industrial effluents (data of initial activity) at TA concentrations of 50 µM TA and not published). higher. In marked contrast the activity measured using 2,6- Given that this chemical interference may take place in DMP showed values close to 90% of the activity measured in identical reaction conditions to those used to measure laccase
  4. 4. 522 M.C. Terrón et al. / Biochimie 86 (2004) 519–522 with ABTS, it may be necessary to examine the likely effects [3] R. Casa, A. D’Annibale, F. Pieruccetti, S.R. Stazi, G. Giovannozzi of these substances on ABTS and other substrates, when Sermanni, B. Lo Cascio, Reduction of the phenolic components in olive-mill wastewater by an enzymatic treatment and its impact on experimentally determining laccase activity. Moreover, as durum wheat (Triticum durum Desf.) germinability, Chemosphere 50 the results presented here suggest, it may be necessary to use (2003) 959–966. more stable substrates such as 2,6-DMP when determining [4] E. Abalulla, T. Tzanov, S. Costa, K.H. Robra, A. Cavaco-Paulo, laccase activity, if the samples are suspected to contain this G.M. Gubitz, Decolorization and detoxification of textile dyes with a laccase from Trametes hirsuta, Appl. Environ. Microbiol. 66 (2000) kind of interfering molecules. 3357–3362. It is important to bear in mind the interference of TA and [5] M.A. Ullah, C.T. Bedford, C.S. Evans, Reactions of pentachlorophe- TA like molecules with laccase activity determinations; nol with laccase from Coriolus versicolor, Appl. Microbiol. Biotech- nol. 53 (2000) 230–234. when studying laccase production in different microorgan- [6] M.A. Pickard, R. Roman, R. Tinoco, R. Vazquez-Duhalt, Polycyclic isms particularly since laccases are often used to detoxify aromatic hydrocarbon metabolism by white rot fungi and oxidation by industrial effluents containing high levels of many different Coriolopsis gallica UAMH 8260 laccase, Appl. Environ. Microbiol. phenolic compounds or strains cultivated in the presence of 65 (1999) 3805–3809. [7] C. Johannes, A. Majcherczyk, Natural mediators in the oxidation of easily oxidized phenolic molecules which are often used as polycyclic aromatic hydrocarbons by laccase mediator systems, Appl. inducers to enhance laccase production. Environ. Microbiol. 66 (2000) 524–528. [8] C.G. Bauer, A. Kühn, N. Gajovic, O. Skorobogatko, P.-J. Holt, N.C. Bruce, A. Makower, C.R. Lowe, F.W. Scheller, New enzyme sensors for morphine and codeine based on morphine dehydrogenase 4. Conclusions and laccase, Fresenius J. Anal. Chem. 364 (1999) 179–183. [9] A.M. Mayer, R.C. Staples, Laccase: new functions for an old enzyme, The results presented in this work, demonstrated that TA Phytochemistry 60 (2002) 551–565. [10] J.M. Harkin, J.R. Obst, Syringaldazine, an effective reagent for could chemically reduce ABTS, thus decreasing laccase- detecting laccase and peroxidase in fungi, Experientia 29 (1973) activity measurements in vitro when laccase is monitored 381–387. using this substrate. Since this chemical reduction takes [11] H. Wariishi, K. Valli, M.H. Gold, Manganese (II) oxidation by man- place in the same conditions used in the laccase detection- ganese peroxidase from the basidiomycete Phanerochaete chrysosporium: kinetic mechanism and role of chelators, J. Biol. assay, our results represent an important warning concerning Chem. 267 (1992) 23688–23695. this commonly used test for measuring laccase activity. [12] B.S. Wolfenden, R.L. Willson, Radical-cations as reference chro- mogens in kinetic studies of one-electron transfer reactions: pulse radiolysis studies of 2,2′-azinobis-(3-ethylbenzthiazoline-6- sulphonate), J. Chem. Soc. Perkin Trans II 1982 (1982) 805–812. Acknowledgements [13] S. Hünig, H. Balli, H. Conrad, A. Schott, Ber zweistufige redoxsys- teme polarographie von 2,2′-Azinen aromatischer, Heterocyclen, Lie- We are grateful to G. del Solar and A.D.W. Dobson for biss Ann. Chem. 676 (1964) 52–65. [14] F. Guillén, A.T. Martínez, M.J. Martínez, Production of hydrogen their critical reading of the manuscript. 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