Enzymatic determination of phenols using peanut peroxidase

Talanta 55 (2001) 1151– 1164 www.elsevier.com/locate/talanta

Enzymatic determination of phenols using peanut peroxidase Nailya A. Bagirova a, Tatyana N. Shekhovtsova a,*, Robert B. van Huystee b b

a Department of Chemistry, Lomonoso6 Moscow State Uni6ersity, B-234, Lenin Hills, 119 899 Moscow, Russia Department of Plant Sciences, Uni6ersity of Western Ontario, 1151 Richmond Street N, London, Ont., Canada N6A 5B7

Received 7 February 2001; received in revised form 30 July 2001; accepted 8 August 2001

Abstract The influence of phenol and its derivatives on the kinetics of oxidation of aryldiamines (indicator-substrates) catalyzed by novel plant peroxidase—cationic peanut peroxidase— was studied. The character of influence of phenols on the kinetics of enzymatic oxidation of benzidine, o-dianisidine, and 3,3%,5,5%-tetramethylbenzidine (TMB) with hydrogen peroxide was found to depend on a correlation between redox properties of phenols and the indicator-substrate of peroxidase. Thus, the catalytic activity of peanut peroxidase is inhibited by phenols with redox potentials higher than that of aryldiamines mentioned above, whereas phenols with potentials below those of aryldiamines, play the role of second substrates of the enzyme. The enzymatic procedures for the determination of numerous phenols on the level of their concentrations 0.05–80 mM were developed using the reactions of benzidine, o-dianisidine, and TMB oxidation. Different analytical signals—the indicator reaction rate and the induction period duration— were used for the determination of phenols, belonging to various groups— the inhibitors and second substrates of the enzyme, respectively. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Peanut peroxidase; Determination of phenols; Aryldiamines oxidation

1. Introduction Data on the application of enzymes isolated from various sources in analytical chemistry show that their sensitivity towards the same substrates [1 – 3] or the action of the same compounds [4 – 9] can differ considerably. A number of papers devoted to the application of enzymes from different origins — mushroom, * Corresponding author. Tel.: +7-95-939-3346; fax: + 795-939-4675. E-mail address: [email protected] (T.N. Shekhovtsova).

yeast, and banana tyrosinases [1], horseradish and Caldariomyces fumago peroxidases [2], horseradish, tobacco, and peanut peroxidases [3] —for the development of enzymatic electrodes for their aromatic substrates determination were published. The application of mushroom tyrosinase [1], horseradish [2] and tobacco [3] peroxidases made it possible to work out the most sensitive analytical procedures for the determination of 4-methylphenol with detection limits (cmin) of 0.15 mmol l − 1 [1] and 0.4 mmol l − 1 [2], and phenol and 4-aminophenol with detection limits of 10 and 0.04 mmol l − 1, respectively [3].

0039-9140/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 1 ) 0 0 5 4 4 - 6

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N.A. Bagiro6a et al. / Talanta 55 (2001) 1151–1164

Ureases from jack beans and soybeans were studied to compare their sensitivity towards an action of copper ions as inhibitor [4]. The biosensor based on urease from jack beans presented higher sensitivity to the copper (II) inhibition action (cmin =13 ng ml − 1). The application of alcoholdehydrogenase from equine liver allowed to develop a more sensitive analytical procedure for the mercury(II) determination (cmin =0.08 mg ml − 1) [5] in comparison to another using the same enzyme from backer’s yeast (cmin =2 mg ml − 1) [6]. The most sensitive procedure for zinc(II) [7] determination is based on its re-activation effect on the apo-enzyme of alkaline phosphatase from Escherichia coli (cmin =0.3 ng ml − 1), but not on apo-enzymes from chicken and Greenland seal intestine. Not so long ago, the systematic comparative investigation of the effect of phenol and its derivatives on the catalytic activity of peroxidases from different origins was initiated [8,9]. The effect of phenols on the activity of horseradish peroxidase isozyme C, alfalfa peroxidase from Medicago Sati6a cell culture, and fungal peroxidase from Phellinus igniarius in the indicator reaction of o-dianisidine oxidation was found to be different. As a result, the sensitivity of the determination of phenols on the level of their concentrations 0.8– 50 mmol l − 1 based on their effect on the indicator-reaction kinetics using peroxidases from various sources was also different, and fungal peroxidase proved to be most sensitive towards the action of phenols. These data show the expedience and importance of investigating the influence of the same compounds on the catalytic activity of enzymes of various origins to reveal the most promising for developing a procedure for chemical analysis. Different sensitivity of the enzymes of diverse sources can be attributed to the different accessibility of the active site, probably due to a difference in the conformation of peptide chains around it. Cationic peanut peroxidase is the first representative of classical plant peroxidases with solved and described structure [10]. A number of papers were dedicated to the investigation of the structure and properties of peanut peroxidase [10–13]. The development of applications of peanut perox-

idase in analytical biochemistry has started rather recently [5]. However, until now a systematic study of substrates and effectors (inhibitors and activators) of this enzyme has not been carried out. Such a systematic investigation is important both from the theoretical and practical points of view, because it can make a valuable contribution to the studies of the substrate specificity and mechanism of the action of peanut peroxidase and may also clarify the prospects of its application in chemical analysis. Thus, the aim of this work was a systematic investigation of the influence of a wide range of phenols on the catalytic activity of peanut peroxidase in different indicator reactions, in order to reveal a correspondence of the character and degree of the action of phenols on the kinetics of indicator processes with their properties and to develop enzymatic procedures for determination of phenols using peanut peroxidase.

2. Experimental

2.1. Reagents Solid preparations of cationic peanut peroxidase isolated from the medium of cultured cells Arachis Hypogea [14] (Rz =2.0) were used. Solutions of peanut peroxidase with concentrations of 1–10 mM were obtained by dissolving enzyme preparations in a Tris–buffer solution (pH 7.5). The exact concentration of a peanut peroxidase solution was determined by spectrophotometry (m405 = 112 mmol − 1 cm − 1 [15]). Solid preparations and solutions of the enzyme were stored in a refrigerator at + 4 °C. Hydrogen peroxide solution (Merck), benzidine, o-dianisidine (3,3%dimethoxybenzidine), 3,3%,5,5%-tetramethylbenzidine (TMB), 2,2%-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), phenol, 2methoxyphenol (guaiacol), 4-aminoantipyrine preparations from Sigma were used. Preparations of other phenols (REAKHIM, Russian Federation) were purified by re-crystallization from rectified ethanol or benzene or by sublimation. Solutions of the enzyme substrates and phenols


were prepared daily by dissolution of accurately weighed amounts in water or rectified ethanol. Solutions with lower concentrations of 2methoxyphenol and 2-bromophenol were prepared by diluting a stock preparations with rectified ethanol. Water was purified on Simplicity system (Millipore) to prepare the aqueous solutions.

2.2. Instrumentation The absorption of solutions was measured on a KFK-2 photocolorimeter (Russian Federation) (l = 2 cm) and a Shimadzu UV-2201 spectrophotometer (Japan) (l =1 cm); pH of buffer solutions and redox potentials of organic compounds were measured by a Econics Expert 001 potentiometer (Russian Federation).

2.3. Procedures 2.3.1. The determination of phenols using peanut peroxidase The rates of enzymatic reactions were monitored by spectrophotometry by an increase in absorbance of solutions due to the formation of colored products. A 7 ml of a buffer solution, the required volumes of a 10 nM peanut peroxidase solution, a solution of phenols with the range of their concentrations of 0.01– 1 mmol l − 1(blank experiments were carried out without adding phenols), a 10 mM substrate solution and water (up to 10 ml total volume of the reaction mixture) were placed sequentially into a glass test-tube with a ground-glass stopper. Finally, the required volume of a 15 mM hydrogen peroxide solution was introduced. At the moment when hydrogen peroxide was added, and the reaction solution was mixed, a stopwatch was started and the absorbance was measured at 15-s intervals for 2– 3 min. The initial rate of the reaction was characterized by a slope (tan h) of the kinetic curve plotted as absorbance versus time. The lower limit of applicable range (cl) was characterized as the minimum determined concentration for n =5 replicate determinations at the confidence level (P) 95% and the relative standard deviation (R.S.D.) 5 33%.

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2.3.2. The measurement of redox potentials of organic compounds Redox potentials of aryldiamines and phenols were measured by a Econics Expert 001 potentiometer using an indicator platinum electrode and a silver/silver chloride reference electrode. Solutions (10 mM) of organic compounds were prepared by dissolving (or diluting) in 20 ml of rectified ethanol and, next, 20 ml of a 0.1 M phthalate buffer solution (pH 5.5) were added.

3. Results and discussions

3.1. The influence of phenols on the kinetics of o-dianisidine oxidation catalyzed by peanut peroxidase The systematic study of the influence of various phenols on the kinetics of o-dianisidine oxidation catalyzed by peanut peroxidase was carried out to continue and expand our previous [8,9] investigations of the influence of phenols on the catalytic activity of peroxidases from different origins. We made an attempt to reveal factors affecting the character of the action of phenols on the kinetics of the indicator process. Phenols with different substitutes (hydroxy-, amino-, nitro-, alkyl-, methoxy-, and halide-groups), in various positions in benzene ring and, as a result, with different acid–base and redox properties, were selected for the investigation. The reaction of o-dianisidine oxidation with hydrogen peroxide was used as indicator (the same reaction was used in our previous works [8,9]). The optimum conditions of o-dianisidine oxidation catalyzed by peanut peroxidase were found previously [16]; concentrations of peanut peroxidase, 0.1 nmol l − 1; o-dianisidine, 0.12 mmol l − 1; hydrogen peroxide, 0.15 mmol l − 1; pH 4.8–5.2, a phthalate buffer solution. The investigation of intermediate and final products and the kinetics of o-dianisidine oxidation catalyzed by peanut peroxidase showed [16] that the reaction proceeded according to the scheme of two-electrons oxidation described for the process catalyzed by horseradish peroxidase [17].

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Scheme 1.

The rate of the enzymatic reaction was monitored by the accumulation of its final product with an orange-brown color (u=465 nm) (Scheme 1). The data of the study of the effect of phenol and its derivatives showed that the rate of the indicator reaction catalyzed by peanut peroxidase decreases in the presence of phenol and resorcinol (Fig. 1, curves 1– 3) proportionally to their concentration (Table 1, by the example of resorcinol). The introduction of pyrocatechol, hydroquinone, and pyrogallol leads to an induction period on kinetic curves (curves 4– 6). The duration of the induction period is directly proportional to the concentration of phenolic compounds (Table 1, by the example of hydroquinone). At the same time, the slope of the second (rising) part of kinetic curves decreases insignificantly in comparison to a blank experiment (in the absence of phenols) and virtually does not change on varying concentration of phenols in the indicator system. Thus, the character of the effect of phenols and their isomers in particular was found to be different. To compare the degree of the effect of studied compounds we used phenols concentrations, at which the degree of inhibition (I, %) was equal to 20% or the induction-period duration was 45 s (depending on the character of the influence of phenols) (Table 2). The degree of inhibition was calculated according to the formula: I (%) = 100%− (tan hinh/tan hblank) 100%, where tan hblank

and tan hinh is the indicator-reaction rate in the absence and in the presence of phenols, respectively. The study of the influence of aminophenols on the catalytic activity of peanut peroxidase showed that 3-aminophenol inhibited the enzyme effectively (Table 2). In the presence of 2- and 4aminophenols, an induction period appears on kinetic curves as in the case of pyrocatechol, hydroquinone and pyrogallol.

Fig. 1. The kinetic curves of o-dianisidine oxidation catalyzed by peanut peroxidase in the absence of phenols (1), in the presence of 10 mM phenol (2), 2 mM resorcinol (3), 2 mM pyrocatehol (4), 2 mM hydroquinone (5) and 2 mM pyrogallol (6). Concentrations of peanut peroxidase, 0.1 nM; o-dianisidine, 0.12 mM; hydrogen peroxide, 0.15 mM; pH 5.0, phthalate buffer solution.

N.A. Bagiro6a et al. / Talanta 55 (2001) 1151–1164 Table 1 Influence of resorcinol and hydroquinone on the kinetics of o-dianisidine oxidation catalyzed by peanut peroxidase (n= 5, P =95%) Phenols

tan h·102

~ind (s)

–

11.690.2

–

2 5 10 2 5 10

9.2 90.1 8.390.1 6.4 9 0.2 11.3 9 0.2 11.49 0.1 11.190.3

Cphenols (mM)

In the absence of phenols Resorcinol

Hydroquinone

– – – 55 93 75 92 115 9 5

The influence of nitrophenols was studied by the example of 2-, 4-nitrophenols and 2,4- and 2,6-dinitrophenols. These compounds did not affect the indicator reaction rate at their concentrations 5 0.1 mmol l − 1. The introduction of

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nitrophenols into the indicator system at their higher concentrations resulted in a considerable increase in the absorbance of the reaction mixture due to the absorbance of nitrophenols. Among the studied alkylphenols, 4-methylphenol at its concentrations 50.1 mmol l − 1 did not affect the rate of o-dianisidine oxidation even after its pre-incubation with the enzyme for 1 h. 2,6-Dimethylphenol and 3,4-dimethylphenol reduced the indicator-reaction rate (Table 2). 2- and 4-methoxyphenols inhibited the enzyme, and 2methoxyphenol was found to be a more effective inhibitor in comparison to its para-isomer. The studied haloid-substituted phenols—4-chlorophenol, 2- and 4-bromophenols and pentabromophenol — inhibited the catalytic activity of peanut peroxidase. The influence of the condensed phenols on the indicator-reaction rate was studied by the example

Table 2 Characteristics of phenols-inhibitors (I) and second substrates (II) of peanut peroxidase in the reaction of oxidation of o-dianisidine and TMB Group

I

Organic compounds

Phenol Pentabromophenol Resorcinol 2-Bromophenol 2-Naphthol 4-Bromophenol 2,6-Dimethylphenol 3-Aminophenol 4-Chlorophenol 2-Methoxyphenol 3,4-Dimethylphenol 4-Methoxyphenol o-Dianisidine Tetramethylbenzidine 1-Naphthol Pyrocatechol 4-Aminophenol 2-Aminophenol Hydroquinone Pyrogallol

II

?K? [19]

10.00 4.43 9.44 8.45 9.63 9.36 10.66 9.87 9.38 9.98 10.38 10.21

[20]

Experimentalb (n =3, P =95%)

o-D

1.089

0.330 9 0.002 0.325 90.004 0.320 90.004 0.315 90.003 0.310 90.002 0.310 90.003 0.305 90.002 0.300 90.001 0.300 90.001 0.295 9 0.002 0.295 90.002 0.290 90.002 0.280 90.003 0.270 90.001 0.270 90.002 0.265 90.003 0.255 90.002 0.255 90.002 0.240 90.004 0.225 90.004

20 35 2 5 3 70 5 0.08 90 0.5 25 2

–c

1.043 –c

1.017 –c –c

0.894 –c

0.868 –c

0.848 0.809 –c

9.85 9.45 10.30 9.71 9.96 9.12

Cphenols (mM)a

EOx/Red (V)

0.797 0.740 0.733 0.730 0.715 0.609

0.8 1.8 1.7 0.8 1.5 0.7

TMB 4 –d

0.5 –d –d –d –d –d –d

2 –d

10

1 2 –d –d

5 1

The concentration of phenols, at which I= 20% (I) and ~ind =45 s (II). The values of redox potentials measured under the same conditions using platinum and silver/silver chloride electrodes. c Data on redox potentials were not found in literature. d An influence has not been studied. a

b

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of 1- and 2-naphthols. The character of their action on the kinetics of the enzymatic process was different; 1-naphthol resulted in the appearance of an induction period on kinetics curves, and 2-naphthol inhibited peanut peroxidase (Table 2). It should be mentioned, that in the presence of 1-naphthol, the first part of kinetic curves had a sharper slope in comparison to that in the case of other phenols affecting the indicator reaction rate by the same type. The slope of a first part of the kinetic curve (tan hI) in the presence of these compounds was (0.89 0.2) ×102 (Fig. 1, curves 4–6). At the same time, tan hI in the presence of 1-naphthol was equal to (3.290.1) × 102. Since the duration of the first part of kinetic curves depends on the concentration of 1-naphthol, we refer to this part of kinetic curves also as an induction period. The slope of the second part almost did not change on varying the concentration of 1-naphthol, as it was in the case of pyrocatechol, hydroquinone, and pyrogallol. So, the obtained results showed that the studied phenolic compounds can be divided into two groups (Table 2) on the basis of the character of their action. 1. Phenols of the first group: inhibitors of peanut peroxidase, they reduce the rate of o-dianisidine oxidation; 2. phenols of the second group: the second substrates of the enzyme, they cause the appearance of an induction period on kinetic curves.

3.2. The reasons of different character of the influence of phenols What are the reasons of such a different character of the influence of phenols belonging to the same class (including isomeric compounds) on the kinetics of the same indicator process? It was supposed previously that the introduction of phenol or its derivatives into the indicator reaction catalyzed by different peroxidases [8] resulted in the combined oxidation of o-dianisidine and the corresponding phenolic compound. So, two enzyme substrates-reductants were present in the indicator system. The mechanism of reactions catalyzed by peroxidases is complicated even in the case of the individual oxidation of a main sub-

strate. In the case of combined oxidation of two substrates the existence of more complicated processes including cross-interaction of intermediate products, may be expected. In such a case, activation or inhibition of the oxidation process of one substrate-reductant by another may be observed [18]. The spectrophotometric study of the kinetics of o-dianisidine oxidation catalyzed by peanut peroxidase in the presence of phenols of the first and second groups— resorcinol and hydroquinone, respectively— was made to examine in details the reasons of different types of the influence of phenolic compounds on the indicator-reaction rate. To observe changes in absorbance in UV spectra for individual and combined oxidation of o-dianisidine and phenols with time, we had to change the ratio of their concentrations in comparison to the optimum conditions of the indicator reactions; the concentration of phenols in this case should be higher than in the case of the indicators like o-dianisidine; because molar absorptivity of o-dianisidine is higher than that of phenols (for example, for o-dianisidine m304 = 18 mmol − 1 cm − 1 and for resorcinol m275 = 2 mmol − 1 cm − 1 [19]). The analysis of the obtained data showed that the absorbance of o-dianisidine at 280–300 nm decreased in the process of its individual enzymatic oxidation (Fig. 2, curves 1 and 2). In the presence of resorcinol, the absorbance at the same wavelength almost did not change (curves 6–8). Resorcinol was not oxidized under the conditions of the indicator reaction; its absorption spectrum remained unchanged for 2 h (curves 3–5). Thus, resorcinol (as well as other phenols of the first group) slows down the oxidation of o-dianisidine and is an inhibitor of peanut peroxidase. In the process of individual oxidation of hydroquinone, its absorbance at 280 nm almost did not change for 10 min, and the absorbance of the forming quinone at 245 nm increased very slowly (Fig. 3, curves 3 and 4). However, the rate of the individual enzymatic oxidation of hydroquinone is too low. If both hydroquinone and o-dianisidine are present in the indicator system, hydroquinone is oxidized at the first stage; one may see a significant increase in the quinone absorbance at 245 nm (curves 5–7). Hence, hy-


Fig. 2. Changes in the absorption spectra in the reaction course of o-dianisidine (1, 2), resorcinol (3 –5) and their combined (6– 8) oxidation catalyzed by peanut peroxidase after mixing the components for 30 s (1, 3, 6), 5 (2, 4, 7), 15 (8), and 120 (5) min. Concentrations of peanut peroxidase, 0.1 nM; o-dianisidine, 20 mM; resorcinol, 0.4 mM; hydrogen peroxide, 0.12 mM.

Fig. 3. Changes in the absorption spectra in the reaction course of o-dianisidine (1, 2), hydroquinone (3, 4) and their combined (5 – 7) oxidation catalyzed by peanut peroxidase after mixing the components for 30 s (1, 3, 5), 5 (2, 6), and 10 (4, 7) min. Concentrations: peanut peroxidase, 0.1 nM; o-dianisidine, 20 mmM; hydroquinone, 0.4 mM; hydrogen peroxide, 0.12 mM.

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droquinone (and, similarly, other phenols of this group) is a second substrate of the enzyme together with o-dianisidine. It should be noted that the oxidation of hydroquinone in the presence of o-dianisidine is faster considerably, than individual hydroquinone oxidation. A similar effect of activation of ferrocyanide oxidation with hydrogen peroxide by o-dianisidine in the presence of horseradish peroxidase was reported [18]. An induction period appeared on kinetic curves of the enzymatic oxidation of o-dianisidine in the presence of ferrocyanide. The appearance of an induction period resulted from the activation of the process of oxidation of a ‘bad’ substrate (ferrocyanide). The activation effect was accounted for the interaction between ferrocyanide and the enzyme–substrate complex (a complex of the peroxidase intermediate form containing one oxidative equivalent (compound II) and a product of one-electron oxidation of o-dianisidine). In our case, the enzymatic oxidation of hydroquinone in the presence of o-dianisidine was accelerated by a factor of 5. Thus, the effect of substrate– substrate activation of phenols of the second group in the presence of o-dianisidine took place in the indicator system. Thus, the scheme of combined enzymatic oxidation of ferrocyanide and o-dianisidine [18] may be used for the description of kinetics of combined oxidation of phenols of the second group and o-dianisidine catalyzed by peanut peroxidase (Scheme 2).

Scheme 2. E is native peroxidase; E1 and E2 are intermediate forms of the enzyme containing two (E1 is Compound I) and one (E2 is Compound II) oxidative equivalents; DH2 is o-dianisidine; E1·DH2 is the complex of Compound I with o-dianisidine E2·DH is the complex of Compound II with semi oxidized o-dianisidine (DH); S is phenolic compound of the second group, P1 P2 are products of individual oxidation of o-dianisidine and phenolic compound, respectively.

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The rate of hydroquinone oxidation in the presence of o-dianisidine catalyzed by horseradish and fungal peroxidases increased by factors 5 and 50, respectively [9]. These data indicated the enzymatic nature of the substrate–substrate activation of the oxidation of phenols in the presence of o-dianisidine and evidenced the similarity of the catalytic properties of peanut and horseradish peroxidases. We made an attempt to find out the dependence of the described character and degree of the influence of phenols on the kinetics of the indicator reaction on some properties of test phenols. The analysis of the data obtained shows that the character of the action of studied phenols on peanut peroxidase does not depend on their acid– base properties, which are quite different; the values of their pKa lie in a wide range from 4.43 for pentabromophenol to 10.66 for 2,6-dimethylphenol (Table 2). However, there is no correlation between the character or degree of the action of phenols on peanut peroxidase and their acid– base properties. It was found that the character of the influence of phenols on the indicator-reaction rate primarily depends on their redox properties. Phenols with potentials lower than that of o-dianisidine are second substrates of peanut peroxidase in the indicator reaction (Table 2). An induction period appears on kinetic curves in the presence of these compounds and its duration is directly proportional to the concentration of phenols (Table 1). The oxidation of phenolic compounds proceeds during the induction period, as it was shown for hydroquinone. Only after the oxidation of that compound is almost complete, the oxidation of o-dianisidine starts, and the solution absorbance increases as the indicator reaction rate is controlled by the formation of oxidation product of o-dianisidine (Fig. 1, curves 4– 6). The slope of the second rising part of kinetic curves in the presence of phenols belonging to the second substrates is not affected by their concentration, but it is somewhat lower than in the absence of phenols. It may be resulted from the consumption of hydrogen peroxide in the oxidation of phenols or the influence of products of oxidation of phenols on the indicatorreaction rate. Phenols with redox potentials higher than that of o-dianisidine (Table 2) are not oxidized in the

reaction and inhibit the catalytic activity of the enzyme. In the presence of these compounds, the indicator-reaction rate decreased directly proportional to their concentration (Table 1). The existing values of redox potentials for a number of phenols were not found (Table 2); therefore, we measured these values by the procedure described in Section 2.3.2. To compare redox properties of all the studied compounds (both phenols and o-dianisidine as well as other peroxidase substrates-indicators mentioned below), their potentials were also measured under the same conditions. These measured values of redox potentials cannot be compared with the existing data [20], because those potentials were measured by the method of potentiometric titration. However, the results obtained are important as they show that the row of the measured and existing values of redox potentials is the same (Table 2). Therefore, these data may be used for the comparison of redox properties of the studied compounds. Amongst the studied compounds, 1-naphthol has potential that is the most similar to that of o-dianisidine. However, in spite of such a small difference in their potentials (D 0.012 V), an induction period appeared on kinetic curves of the indicator reaction. Taking into consideration the similarity of their potentials, one may explain an abrupt slope of the first part of kinetic curves in the presence of 1-naphthol (discussed above) by nearly simultaneous oxidation of 1-naphthol and o-dianisidine. The degree of the influence of phenols on the indicator-reaction kinetics did not correlate with the values of their redox potentials (Table 2). This phenomenon may be a consequence of some other factors, viz. size, positions of substitutes and, apparently, the accessibility of the hydroxyl group of phenolic compounds. Thus, pentabromophenol inhibited peanut peroxidase very weakly; the degree of the inhibitory action was equal to 20% at its concentrations 35 mmol l − 1 and it changed insignificantly with an increase in its concentration. 2,6-Di-tert-butyl-4methylphenol at its concentrations 5 0.1 mmol l − 1 did not affect the kinetics of the enzymatic oxidation of o-dianisidine at all. This fact may be


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Scheme 3.

accounted for the location of two bulky substitutes in the vicinity of the hydroxyl group resulted in sterical hindrances for a compound binding with the enzyme molecule. On the other hand, the size of molecules of the condensed phenols (1- and 2-naphthols) is comparably large, but hydroxyl groups are not shielded, and these compounds affect the indicator-reaction rate at their considerably low concentrations (Table 2). However, it should be mentioned that phenol itself is one of the weakest inhibitors of peanut peroxidase (Table 2) despite a small size of its molecule and the presence of an unshielded hydroxyl group. At the same time, the availability of a single functional group may be supposed to be insufficient for the effective binding of phenol with peanut peroxidase globule and, therefore, for the competition with o-dianisidine.

Peroxidases catalyze the oxidation of a wide range of organic and inorganic compounds. Phenol and guaiacol (2-methoxyphenol) belong to the studied class of compounds. We also found that the specific activity of peanut peroxidase towards these compounds and ABTS is much lower in comparison to the enzyme activity towards o-dianisidine, 12, 13, 20 and 700 U mg − 1, respectively (the specific activity of peanut peroxidase was measured using procedures [21–24]). Hence, the application of these compounds as indicator-substrates of peanut peroxidase was inexpedient. Benzidine (EOx/Red = 0.31090.004 V) and TMB (EOx/Red = 0.27090.001 V) were selected as indicators for the investigation. The specific activity of the enzyme towards these substrates (measured using procedures [24,25]) is 750 and 400 U mg − 1, respectively.

3.3. The selection of indicator systems

3.4. The influence of phenols on the kinetics of 3,3 %,5,5 %-tetramethylbenzidine oxidation catalyzed by peanut peroxidase

To confirm the advanced hypothesis on the dependence of the character of the influence of phenols on the correlation between their redox potentials and those of o-dianisidine we studied the effect of the same phenols on the kinetics of oxidation of other peroxidase substrates-indicators with various structures, absorption-band parameters, and redox potentials (which is the primary factor).

The results of a comparative spectrophotometric study of the intermediate and final products and the kinetics of TMB oxidation catalyzed by peanut and horseradish peroxidases allowed us to assume that TMB oxidizes in the presence of both peroxidases according to the scheme analogous to the one-electron oxidation with a formation of free cation-radicals [16,25] (Scheme 3).

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The rate of the indicator reaction was monitored by the accumulation of the final product of enzymatic oxidation of TMB with a yellow color (u = 465 nm). To optimize the conditions of TMB oxidation with hydrogen peroxide in the presence of peanut peroxidase, we studied the indicator-reaction rate as a function of pH and concentrations of TMB and hydrogen peroxide. The optimum conditions were as follows, concentrations of peanut peroxidase, 0.2 nmol l − 1; TMB, 0.15 mmol l − 1; hydrogen peroxide, 0.3 mmol l − 1; pH 6.0 – 7.0, a Tris–buffer solution. Phenols with redox potentials higher than that of TMB decreased the indicator-reaction rate (Table 2). In the presence of phenols with redox potentials lower than that of TMB, two parts on kinetic curves of TMB oxidation were observed. The duration of the first (slightly sloping) part (the induction period) was proportional to the concentration of the phenolic compound in the indicator system.

Fig. 4. Changes in the absorption spectra in the reaction course of TMB (1 – 3), hydroquinone (4 – 6) and their combined (7 –10) oxidation catalyzed by peanut peroxidase after mixing the components for 30 s (1, 4, 7), 3 (8), 5 (2, 5, 9), and 10 (3, 6, 10) min. Concentrations: peanut peroxidase, 0.2 nM; TMB, 25 mM; hydroquinone, 0.4 mM; hydrogen peroxide, 0.3 mM.

A spectrophotometric study of the individual and combined oxidation of hydroquinone and TMB showed (Fig. 4) that despite different mechanisms of the oxidation of o-dianisidine and TMB catalyzed by peroxidase (Schemes 1 and 3), these substrates activated the oxidation of the studied phenols with lower redox potentials. Thus, almost the same phenomenon as that in the reaction of o-dianisidine oxidation was observed in the case of the influence of the studied phenols on the rate of enzymatic oxidation of TMB, and these data confirmed the advanced hypothesis. 3.5. The influence of phenols on the kinetics of benzidine oxidation catalyzed by peanut peroxidase Benzidine is used as the peroxidase indicatorsubstrate much more seldom, than its derivatives like o-dianisidine, o-tolydine and TMB. However, benzidine was chosen for our subsequent investigation and confirmation of the advanced hypothesis, because its redox potential is higher than that of previously studied substrates. The study of the mechanism of benzidine oxidation catalyzed by horseradish peroxidase has shown that a single product with a blue color with two absorption maxima (410 and 595 nm) [26] is formed. We assumed that the meriquinone complex is formed as a result of quinone diimine association with original diamine [22] (Scheme 4). A product with two maxima at 380–420 and 590–620 nm was formed as a result of benzidine oxidation catalyzed by peanut peroxidase as well as in the case of horseradish peroxidase. The positions of maxima of the product of the enzymatic oxidation of benzidine were similar to those of absorption maxima of the intermediate products of oxidation of its derivatives, o-dianisidine and TMB (Schemes 1 and 3). However, in this case the meriquinone complex was the single product of benzidine oxidation. The optimum conditions for benzidine oxidation catalyzed by peanut peroxidase were as follows, concentrations of peanut peroxidase, 0.1 nmol l − 1; benzidine, 0.1 mmol l − 1; hydrogen peroxide, 0.3 mmol l − 1; pH 4.8– 5.2, phthalate buffer solution (u= 400 nm).


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Scheme 4.

Resorcinol at its concentrations of 0.3 mmol l − 1 reduced the rate of the enzymatic oxidation of benzidine by 20%. The duration of the induction period on the kinetic curves was equal to 45 s in the presence of pyrogallol at its concentrations of 1 mmol l − 1. The most explicit confirmation of the hypothesis was obtained using 3-aminophenol and 2- and 4-methoxyphenol. These compounds decrease the rate of o-dianisidine oxidation since their redox potentials are higher than that of o-dianisidine (Table 2). The character of the influence of these phenols changed in the reaction of benzidine oxidation: kinetic curves of this reaction had two parts. In the presence of 3-aminophenol (Fig. 5) and 2-methoxyphenol, a slightly increasing part of kinetic curves appeared at their concentrations of ]0.5 and 0.8 mmol l − 1, respectively. The slopes of both parts diminished with an increase in the concentrations of these compounds. The length of the first part did not change with an increase in the concentration of phenols and was equal to 12095 and 609 5 s, respectively. The length of the first part of the kinetic curve was equal to 45 s when 4-methoxyphenol was introduced in the indicator system at its concentrations of 1 mmol l − 1. And in the case of 4-methoxyphenol, the length of the first part of kinetic curve increased and the slope of the second part diminished proportionally to its concentration. Such a character of kinetic curves probably indicates that the oxidation of phenols proceeds in the indicator system at the first stage. Thus, these compounds play the role of second substrates of peanut peroxidase. The values of potentials of

these phenols are close to the potential of benzidine, the character of the action of phenols is comparable to the type of inhibition (as in the case of 3-aminophenol). A decrease in the rate of benzidine oxidation owing to the action of the products of enzymatic oxidation of phenols may lead to a decrease in the slope of the rising part of kinetic curves with an increase in the concentrations of 3-aminophenol, methoxyphenols and pyrogallol. Therefore, the results obtained show that the character of the influence of phenols on the kinetics of benzidine oxidation depends also on the correlation between redox potentials of phenols and the enzyme substrate, as it was in the case of oxidation of benzidine derivatives.

Fig. 5. The kinetic curves of benzidine oxidation catalyzed by peanut peroxidase in the absence (1) and presence of 3aminophenol (2, 3). Concentrations: peanut peroxidase, 0.1 nM; benzidine, 0.1 mM; 3-aminophenol, 1 (2) and 7.5 (3) mM; hydrogen peroxide, 0.3 mM; pH 5.0, phthalate buffer solution.

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Table 3 Analytical characteristics of the procedures for the determination of phenols using peanut peroxidase in the oxidation reaction of aryldiamines Phenols

3-Aminophenol 2-Methoxyphenol 4-Methoxyphenol 1-Naphthol Pyrocatehol Hydroquinone Pyrogallol

a

3,3%,5,5%-Tetramethylbenzidine

Benzidine Applicable range (mM); calibration graph equation

Cl (mM); R.S.D. (%)

0.1–6 6=10.22–1.21x 0.5–10 6= 11.77–0.81x 0.8–10

0.1 4 0.5 5 0.8

6= 11.31–0.68x 0.7–10

3 0.7

~= 34.43+12.33x

4

Applicable range (mM); calibration graph equation

Cla (m?); R.S.D.b (%)

Applicable range (mM); calibration graph equation

1–10 6=10.74–0.45x 0.05–6 6 =7.61–2.19x 0.3–3

1 19 0.05 1 0.3

0.2–7 6 =11.78–1.14x

6 =9.83–1.87x 1–15

12 1

5–80

6 =10.51–0.31x 0.5–5 ~ = 23.41+25.52x 3–30 ~ = 17.24+3.15x 1–10 ~ =32.86+8.54x 0.5–5 ~= 15.38+23.87x

4 0.5 20 3 20 1 22 0.5 15

6 = 9.71–0.18x 1–10 ~ =40.28+9.59x 5–50 ~= 12.499+4.11x 5–50 ~ =34.86+1.74xx 0.5–50 ~ =24.08+19.35x

Cl (mM); R.S.D. (%) 0.2 3

–c

–c

2 4 1 3 5 11 5 16 0.5 4

The lower determination limit (lower limit of applicable range). R.S.D. for phenols at their concentration equal to Cl. c The procedures have not been developed. 6, The indicator reaction rate (tan a102); ~, the duration of the induction period; x, concentration of phenols, mM. b

c

c

c

0.8–8 ~= 30.68+15.02x

0.8 3


Resorcinol

o-Dianisidine

N.A. Bagiro6a et al. / Talanta 55 (2001) 1151–1164 Table 4 The results of the determination of phenols at their combined presence using the reaction of o-dianisidine oxidation (n= 5, ? = 95%) Phenols –

Resorcinol Hydroquinone Resorcinol + Hydroquinone Resorcinol + Hydroquinone 1-Naphthol 2-Naphthol 1-Naphthol + 2-Naphthol 1-Naphthol + 2-Naphthol

C (mM)

tan h·102

~ind (s)

–

10.890.4

4 12 2 4 (2:1) 2 12 (6:1) 2 1 2 10 1 (1:2) 2 1 (1:10) 10

8.190.1 5.7 9 0.2 9.5 9 0.2

– – 42 93

8.0 9 0.2

40 9 1

5.9 9 0.1

38 92

10.5 9 0.2 8.3 9 0.2 5.19 0.2

51 94 – –

8.6 9 0.3

48 92

4.8 9 0.2

469 2

–

3.6. The determination of phenols using peanut peroxidase The analytical procedures for the determination of a number of phenols were developed using indicator reactions of benzidine, o-dianisidine, and TMB oxidation catalyzed by peanut peroxidase. Different analytical signals—the indicator-reaction rate and the induction period duration— were used for the determination of phenols-inhibitors and second substrates, respectively. The applicable concentration ranges of phenols were found under the optimum conditions for their most efficient influence in every indicator reaction mentioned above. The calibration graphs were plotted as the indicator-reaction rate (tan h) versus the concentration of inhibitor phenols or the induction-period duration (~ind, s) versus concentration of phenols being second substrates. Table 3 shows that the procedures using o-dianisidine as peanut peroxidase substrate have the highest sensitivity of the determination of the majority of phenols.

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The comparison of analytical characteristics of the developed procedures using the reaction of o-dianisidine oxidation catalyzed by peanut peroxidase and horseradish, alfalfa, and fungal peroxidases [8] showed that the application of peanut peroxidase provided the determination of phenols with the highest sensitivity. Only fungal peroxidase was more (or equally) sensitive towards hydroquinone (or pyrogallol) action. For instance, the lower determination limits for resorcinol using peanut, fungal, horseradish, and alfalfa peroxidases are equal to 1, 3.2, 5.4 and 13 mmol l − 1; for hydroquinone the values are 1, 0.8, 4.5 and 7.2 mmol l − 1, respectively. The comparison of the obtained results with the sensitivity of enzymatic electrodes for the determination of phenols [1–3] showed that the developed procedures are more sensitive only in comparison to tyrosinase biosensor [1]. As the character of the effect of phenols on the kinetics of the indicator reactions was different, and different analytical signals were used for their determination, it was expedient to study the possibility of the individual determination of isomers of different groups at their combined presence. By the example of two pairs of isomers, it was shown that it is possible to determine individually resorcinol and hydroquinone, 1- and 2-naphthols at their combined presence at their concentration ratios of 2:1–6:1 and 1:2–1:10, respectively (Table 4) using the indicator reaction of o-dianisidine oxidation.

4. Conclusions As a result of a systematic investigation of the influence of a wide range of phenols on the kinetics of the oxidation of benzidine, o-dianisidine, and TMB catalyzed by peanut peroxidase, the studied phenols were divided into two groups—the inhibitors and second substrates of peanut peroxidase. The found character of the influence of phenols does not depend on their acid–base properties, but depends primary on the correlation between the values of redox potentials of phenols and the main peroxidase substrate-reductant— benzidine or its derivatives.

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The effect of the substrate– substrate activation was observed in the case of simultaneous presence of two substrates in the indicator system; benzidine, o-dianisidine, and TMB activate the enzymatic oxidation of phenols with redox potentials lower than those of aryldiamines. This effect leads to the appearance of an induction period on kinetic curves. The application of different substrates and analytical signals—the indicator reaction rate and the induction period duration—for the development of analytical procedures for the determination of phenols that are the inhibitors and second substrates, makes it possible to regulate the sensitivity and selectivity of the determination of some phenols in the presence of others.

Acknowledgements This study was partially supported by the Russian Foundation for the Basic Research grant N 00-03-32548a.

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