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 Laccases (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) are multi-copper phenoloxidases detected in many plants and secreted by numerous fungi. They catalyse the oxidation of a number of quite different aromatic substances (diphenols, methoxy-substituted monophenols, aromatic amines) using oxygen as the final electron acceptor [1,2]. Laccases from some basidiomycetous fungi have been shown to be effective in oxidizing a number of pollutants as
Abbreviations: ABTS, 2-2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid); TA, Tannic acid; 2,6-DMP, 2,6-dimethoxyphenol. * Corresponding author. Tel.: +34-91-8373112 Ext. 4413; fax: +34-91-5360432. E-mail address: aldo@cib.csic.es (A.E. González). 1 INIA, Carretera de la Coruña Km 7.5, 28040 Madrid, Spain 2 GBF-National Research Centre for Biotechnology, D-38124 Braunschweig, Germany 3 Universidad Politécnica de Pachuca, Zenpoala. C.P. 43830. Estado de Hidalgo, México 4 Instituto Cubano de Derivados de la Caña de Azúcar, Havana, Cuba 0300-9084/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2004.07.013
well as low molecular weight toxic phenols [3], industrial dyes [4], chlorophenols [5], together with anthracene, phenanthrene and other polycyclic aromatic hydrocarbons (PAHs) [6,7]. The rather broad substrate specificity of fungal laccases has generated an increased interest in a variety of different biotechnological applications for this methalloenzyme. Currently laccases are used in pulp delignification, textile dye bleaching, bioremediation and effluent detoxification, as well as being used in detergents and in biosensors, among other applications [8,9]. Laccase activity is commonly determined spectrophotometrically based on the capacity of this enzyme to oxidize– colorize specific aromatic compounds such as guaiacol, syringaldazine [10], or 2,6-dimethoxyphenol (2,6-DMP) [11]. One of the most commonly used substrates is the electronrich non-phenolic compound 2-2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) [12], because, in contrast to the phenolic substrates forming quinones, the oxidation potential of ABTS is not pH-dependent within the range 2–11 [13] and proceeds in one step. Oxidation of ABTS by laccase results in the production of a green–blue coloured radical cation (ABTS+•) measurable
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at 436 nm (eo= 29300 M–1 cm–1). The reaction mixture usually consists of acetate buffer at around pH 5, ABTS as the substrate (10 mM final concentration), and the fungal extracellular medium containing the laccase activity to be measured. In this work, we demonstrate that under these reaction conditions but in the absence of any enzyme, tannic acid (TA) is able to carry out the chemical reduction of ABTS+•. This results in an underestimation of laccase activity values determined by this method in biological samples containing TA or related aromatic compounds.
as described above but without the addition of TA. Laccase activity was measured immediately after the addition of TA solutions to enzymatic crudes using both, ABTS and 2,6DMP as substrates.
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 spectrophotometer from 400 to 500 nm. Different ABTS spectra were carried out in the absence and in the presence of TA (0.25, 0.50, and 1 µM final concentrations) or sodium ascorbate (25 µM).
TA (Tannic acid powder pure, USP, empirical formula C76H52O46), was obtained from Merck (Darmstadt, Germany). ABTS was purchased from Boehringer-Mannheim (Germany) and 2,6-DMP from Fluka (Germany). All other chemicals were reagent grade obtained from Merck or Sigma-Aldrich.
2.4. Laccase activity determination Laccase activity was measured, using 2,6-DMP [11] or ABTS [12] as enzyme substrates. One unit of laccase activity is defined as the formation of 1 µmol of product per min. All assays were performed in duplicate using a Shimadzu UV1603 spectrophotometer.
3. Results and discussion 2.2. Organism and maintenance Basidiomycetes Coriolopsis gallica (A-241) and Trametes sp. I-62 (B-24), were obtained from the IJFM (Instituto Jaime Ferrán de Microbiología) collection. The fungal cultures were maintained on malt agar slants (2% malt extract, 2% Bacto Agar), grown for 10 days at 28 °C, and stored at 4 °C. 2.3. Culture conditions C. gallica was grown on agar plates with modified Czapeck medium [14] for seven days at 28 °C. Ten plugs (1 cm2) were cut and inoculated under sterile conditions into 500 ml culture flasks containing 300 ml of Kirk growth medium with 0.4 mM veratryl alcohol [15]. After incubation for 48 h at 28 °C in an orbital shaker (200 rpm), an inoculum of 1:10 (v/v) was transferred into 250 ml Erlenmeyer flasks containing 100 ml of the same medium. Samples were incubated at 28 °C and 125 rpm for 7 days. Afterwards, a filter-sterilized (0.22 µm) water-solution of 50 mM TA was added to the culture medium to reach a final concentration of 200 µM, and the cultures were incubated under the same conditions for eight more days. During this time laccase activity was measured daily in the extracellular fluids using ABTS and 2,6DMP as substrates. Controls without TA were also run and monitored daily. The assays to determine laccase activity in the presence of different concentrations of TA (0, 0.1, 1, 5, 9, 20, 50, 100 and 200 µM, final concentrations) were performed by mixing them with extracellular fluids from 12-days-old cultures of Trametes sp. I-62, grown under the same culture conditions
The interference in the laccase-detection assay using ABTS as the substrate (evidenced by the decrease of absorbance at 436 nm in the presence of different industrial effluents), is a frequent observation in our laboratory (data not published). Given that all these wastewaters come from industries that use plants as raw material, we speculated that the common substances which may be responsible for this interference could be polyphenolic based compounds; which in addition are molecules which could be easily oxidized by the enzyme. Thus, we determined the total phenol and tannin content in some of these effluents, which indicated that tannins represent a significant percentage of their phenolic composition [16]. For this reason, TA (the most abundant polyphenolic molecule in plants after lignin) was selected as a model compound in our study. Experiments were performed initially to elucidate whether the observed interference with laccase activity might be due to a direct interference with the laccase enzyme itself, or to a chemical interference with ABTS. With this in mind, we monitored laccase activity in C. gallica over an 8-day period; grown in either the presence or absence of 200 µM of TA. Laccase activity was measured using ABTS or 2,6-DMP (Fig. 1). Significant differences were observed with much higher laccase activity values being detected in the presence of TA using 2,6-DMP as substrate as opposed to ABTS. For example, laccase activity determined with 2,6DMP was 3.2-fold higher than in the control without TA, in day 6 samples. On the other hand, laccase activity determined with ABTS was 9-fold lower than that observed on the same day in the control. These results suggested the possible interference of tannic acid with the ABTS molecule. This is
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Fig. 1. Laccase activity determined as oxidation of (n) 2,6-DMP or (•) ABTS in the extracellular fluid of Coriolopsis gallica. Each point represents: [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 coworkers [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 solution of ABTS both in the presence and absence of different concentrations of TA were then assessed (Fig. 2A). The absorbance values of ABTS were markedly lower especially in the 400–460 nm range, at increased concentrations of TA. Moreover at TA concentrations of 0.5 µM and higher, the characteristic shoulder around 420 nm was absent. The spectrum of ABTS in the presence of 25 µM ascorbate, a wellknown chemical ABTS reducer [12], was also performed; and were shown to be very similar to those of ABTS in the presence of 0.5 and 1 µM TA (Fig. 2A). This strongly suggests that TA can chemically reduce the ABTS molecule. It is well established that at ABTS concentrations greater than 1 mM, solutions appear green-blue but become colourless when reducing agents such as ascorbate or cysteamine are added, even at relatively low concentrations [12]. A similar change in colour was also observed here when TA was added to ABTS. In addition we performed assays on a native laccase enzyme crude from an 12-day-old culture of Trametes sp. I-62, in order to verify whether TA–ABTS interference also affected enzymatic activity measurements. Laccase activity was measured both in the presence and absence of different concentrations of TA and using either ABTS or 2,6-DMP as substrates (Fig. 2B). The results obtained indicated a dramatic decrease in ABTS mediated laccase activity following addition of 5 µM TA, reaching minimum values (less that 5% of initial activity) at TA concentrations of 50 µM TA and higher. In marked contrast the activity measured using 2,6DMP showed values close to 90% of the activity measured in
Fig. 2. (A) Optical absorption spectra of ABTS in the absence (a) and in the presence of different concentrations of TA (0.25 (b), 0.50 (c), and 1 µM (d)) or sodium ascorbate (25 µM) (e). (B) Laccase activity determined using (n) 2,6-DMP or (•) ABTS in the presence of different concentrations of TA. For the experiment, extracellular fluids containing high laccase activity from 12-days old cultures of Trametes sp. I-62 were used.
the absence of TA, even in the presence of the highest TA concentration assayed (200 µM). Recently, other organic compounds have also been reported to be capable of reducing ABTS. Johannes and Majcherczyk [18] have demonstrated that several sulfhydryl organic compounds, described as laccase inhibitors, are not true inhibitors but rather are substances which are able to chemically reduce the coloured radical cation ABTS+• to ABTS, resulting in the decolourising of the solution. The reduction of ABTS by some physiological organic acids and a phenolic lignin-related compound has also been described [19]. The results presented here indicate that TA is also capable of chemically reducing ABTS+• in reaction conditions commonly used to detect laccase activity, leading to the possibility that much lower laccase activity values will be obtained when this compound is present in the reaction mixture. This interference could probably be extended to include other easily oxidised structural-related phenolic compounds; and may explain at least in part, the decrease in absorbance observed in the presence of some industrial effluents (data not published). Given that this chemical interference may take place in identical reaction conditions to those used to measure laccase
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with ABTS, it may be necessary to examine the likely effects of these substances on ABTS and other substrates, when experimentally determining laccase activity. Moreover, as the results presented here suggest, it may be necessary to use more stable substrates such as 2,6-DMP when determining laccase activity, if the samples are suspected to contain this kind of interfering molecules. It is important to bear in mind the interference of TA and TA like molecules with laccase activity determinations; when studying laccase production in different microorganisms particularly since laccases are often used to detoxify industrial effluents containing high levels of many different phenolic compounds or strains cultivated in the presence of easily oxidized phenolic molecules which are often used as inducers to enhance laccase production.
[3]
[4]
[5]
[6]
[7]
[8]
4. Conclusions [9]
The results presented in this work, demonstrated that TA could chemically reduce ABTS, thus decreasing laccaseactivity measurements in vitro when laccase is monitored using this substrate. Since this chemical reduction takes place in the same conditions used in the laccase detectionassay, our results represent an important warning concerning this commonly used test for measuring laccase activity.
Acknowledgements We are grateful to G. del Solar and A.D.W. Dobson for their critical reading of the manuscript. This work was supported by the CICYT (Madrid, Spain) BIO 97-0655 and Comunidad de Madrid (CAM 07M/0730/1997). J.M. Carbajo and M.C. Terrón acknowledge support from pre- and postdoctoral grants, from Conserjería de Educación y Cultura de la Comunidad Autónoma de Madrid (Spain).
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