Biochi:nie (1992) 74, 143-148 © Soci6t6 franqaise de biochimie et biologie mol6culaire / Elsevier, Paris
143
Oxidation of hydroquinone by both cellular and extracellular grapevine peroxidase fractions JM Zapata, AA Cald6ron, R Mufioz, A Ros Barcel6* Department of Plant Biology (Plant Physiology). University of Murcia, Campus of Espinardo. E-30071 Mmz'ia. Spain (Received 11 March 1991; accepted 5 December 1991)
Summary m The oxidation of hydroquinone by two peroxidasc (EC 1.1 I. !.7) fractions obtained from the cells and spent medium of cell cultures of grapevine (Vitis vin[ft,racv Monastrell) has been studied, and their comparative efficacy (kJKM ratio) studied in both the H,,O.,-consuming and hydroquinone-consuming reactions. While the efficacy in the H,,O2-consuming reaction is practically identical for both enzyme fractions, the cellular peroxidase has five-fold more efficacy in the hydroquinone-consuming reaction than the peroxidase located in the spent medium. Screening of cellular peroxidases capable of oxidizing hydroquinone on polyacrylamide gels, by means of a staining reaction based on the nucleophilic attack of 4-aminoantipyrine on p-benzoquinone in acidic media, reveals that all the cellular peroxidase isoenzymes are capable of oxidizing hydroquinone, probably yielding a quinone-diimine as a product of the staining reaction. Since isoperoxidases found in cellular fractions are also present in the spent medium, the values found for the different efficacies in the hydroquinone-consuming reaction must be considered as the results of the different proportions in which each peroxidase isoenzyme was found in the two fractions. The localization of a benzoquinone-generating system of high efficacy inside the plant cell, and probably located in vacuoles, is discussed with respect to the harmful role which the quinone/semiquinone pair might play in cell death, as part of the hypersensitive response expressed within the mechanism of plant disease resistance. peroxidase/hydroquinone oxidation/benzoquinone/grapevine Introduction Phenolic compounds form one of the most prominent classes of natural products in plants. They are ,~ery reactive chemically and are easily subjected to oxidation, substitution, and coupling reactions [1]. The degradation of p-hydroxybenzoic acids has been especially investigated in connection with decarboxylation and O-demethylation reactions [ 1]. Thus, the metabolism of p-hydroxybenzoic acids constitutes a wellknown route to simple phenols, the formation of hydroquinones being a good example. The hydroxylation-induced decarboxylation of p-hydroxybenzoic acids to hydroquinones is catalysed by peroxidases both in in vitro and in vivo conditions [21, using stoichiometric amounts of H:O2. If H_,O2 is present in stoichiometric amounts, hydroquinones are considered as end products of these catabolic reactions, and can be further glucosylated through glucosyltransferases to monoglucosides [3]. Arbutin, the monoglucoside of hydroquinone, is the
Abbreviations: AAE 4-aminoantipyrine; BQ, p-benzoquinone; HQ, hydroquinone; MN, 4-methoxy-o~-naphthol *Correspondence and reprints
principal active component of Arctostaphylos uvaursi, and is used as a urethral disinfectant, and a potent suppressor of the synthesis of melanins in animals 131. When H:O: is present in excess, hydroquinones are oxidized by peroxidases to the semiquinone radical, which is very unstable and rapidly transformed to paraquinones, dimers or polymers, depending on the substitution pattern. In this reaction, superoxide is generated, since the semiquinone radical intermediate has a high affinity for oxygen [4]. Superoxide radicals can further react with hydrogen peroxide, formed through the enzymatic or spontaneous dismutation of the superoxide radical, to form even more damaging species of oxygen, such as hydroxyl radicals and singlet oxygen [5]. In view of the multiple deleterious effects of semiquinones on plant cells, the oxidation of hydroquinone-type phenols by peroxidase would seem to be of great importance during aging stages 131 and, therefore, during the hypersensitive necrotic reaction of grapevines shown in response to fungal attack, as part of the mechanism of disease resistance I6]. In this report, we study the capacity of a cellular and a spent medium peroxidase fraction obtained from
144 grapevine cell cultures to oxidize hydroquinone (HQ) to p-benzoquinone (BQ). Further, a zymographic method is developed to screen grapevine peroxidase isoenzymes capable of oxidizing HQ through the coupling properties of the p-benzoquinone end-product with 4-aminoantipyrine (AAP). Finally, the possible participation of cellular peroxidases, through the oxidation of hydroquinone, in the previously described hypersensitive (necrotic) reaction in grapevines is discussed.
Materials and methods Reagents Hydroquinone was obtained from BDH Chemicals Ltd (Dorset, UK) and p-benzoquinone from Fluka Chemie AG (Buchs, Switzerland). Guaiacol and H20, were obtained from Merck (Darmstadt, Germany) and 4-methoxy-ot-naphthol from Aldrich Chemie (Steinheim, Germany). Ampholines and 4-aminoantipyrine were obtained from Sigma Chemical Co (St Louis, MO, USA). All other chemicals used in this work, including plant cell culture reagents, were the best available commercial products.
Materials Callus cultures were obtained from immature pericarp tissue of Monastrell grapes (Vitis vinifera L cv Monastreli) of approximately 5 mm diameter. The culture medium, based on that of Gamborg et a! [7], was supplemented with casein hydrolysate (250 mg/1), kinetin (0.2 ml/l), or-naphthalene acetic acid (0.1 mg/1) and Morel's vitamins [8]. Cultures were maintained in the dark and subcultured at approximately three-week intervals. Starting from friable callus subcultured for 24 months, grape cells were cultured in suspension on the above described culture medium in 100 ml flasks with orbital shaking (125 rpm). Suspension cultured cells were grown at 25°C in darkness for 15 days. Peroxidase from the spent medium of the cell culture was obtained by filtration of the cells through a Btichner funnel. Peroxidase from the cells was obtained by homogenization of the cells recovered in the filter in an Omni-mixer mechanical blade in the presence of 0.1 M Na-Mes buffer at pH 6,5 and 2.5c/~ (w/v~ sucrose, and centrifugation as already described 191. Both peroxidase fractions were dialyzed against 50 mM Tris-HCI buffer at pH 7.5 and stored at -20°C.
Kinetic assays Unless otherwise noted, the spectrophot~netric assay was performed at 25"C in a 1.0-ml assay volume, containing 7.5 mM hydroquinone, 0.5 mM H,,O.,, and 100 mM Tris-acetate buffer at pH 5.0. After mixing, the enzymatic reaction was initiated by adding 25 ~tl of enzyme. The increase in absorbance at 250 nm over a time period of ! rain was measured, the absorbance increase always being linear with respect to time. The oxidation rate was expressed as nmol of product formed min-I using a e,~, = i.9 x ilM M-I cm-I for p-benzoquinone [10l. The assay of peroxidase activity using 4-methoxy-o~-naphthol as substrate was carried out as described in [ ! I l, and the enzyme amounts tin nkat) in the assays were calculated using this substrate.
Zymographic assays Isoelectric focusing of grapevine peroxidase isoenzymes was performed on 3.5-10.0 pH gradients as described elsewhere [! 2]. Staining with 4-methoxy-ot-naphthol as subs(rate was carded out as previously described [11 ]. The reaction of peroxidase isoenzymes with hydroquinone was demonstrated using an incubation medium identical to that described above in the kinetic assay. The reaction was performed at 25°C for 30 min. After removal of the hydroquinone-containing reaction media, the staining reactian was developed by adding 50 mM of 4aminoantipyrine in 0. l M HCI. The red-wine colour of the product was seen l0 min after the initiation of the coupling reaction, and was stable for at least 0.5 h. Densitometric recording of 4-methoxy-ot-naphthol stained isoenzymes was carried out as described I! II, using a JoyceLoebi MK It scanner densitometer.
Results Characterization of the oxidation of hydroquinone by two grapevine peroxidase fractions The oxidation of hydroquinone (HQ) by peroxidases can be followed spectrophotometrically, since the pbenzoquinone (BQ) end-product has a characteristic absorption band in the region 2 3 5 - 2 6 8 [10]. Thus, oxidation of hydroquinone by grapevine peroxidases can be monitored by the increase of absorbance at 250 nm, which is accompanied by a decrease in the absorbance at 290 nm of the reaction media over time (fig I A). The appearance o f an isosbestic point at X = 268 nm in the consecutive spectra o f the reaction media for high reaction times or high e n z y m e concentrations seemed to suggest that there was a constant transformation of HQ to BQ, the two species (substrate and product) being kinetically correlated. The formation of BQ in these reaction media was strictly dependent on the addition of H202. However, a certain chemical oxidation took place in the absence of e n z y m e (fig IB). These observations, together with the inability of grapevine catechol oxidase (o-diphenol: 02 oxido~eductase, EC 1.10.3.1) to oxidize HQ [13], and the fact that it was not possible to determine laccase (pdiphenol:O2 oxidoreductase, EC 1.10.3.2) activity in grapevine protein fractions by means of the syringaldazine test [14], suggest that the oxidation o f HQ to BQ must be attributed to peroxidase activities. In order to characterize the peroxidatic oxidation of HQ by cellular and spent m e d i u m peroxidases, the oxidation rate (estimated from the increases in absorbance at 250 nm) was e x a m i n e d as a function o f the pH, and of both n202 and HQ concentration. Variations in the pH o f a 100 m M Tris-acetate buffer in the 3.0-7.0 range did not drastically modify the HQ oxidation rate by either cellular or spent m e d i u m peroxidase activities (fig 2), although an
145
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143
Oxidation of hydroquinone by both cellular and extracellular grapevine peroxidase fractions JM Zapata, AA Cald6ron, R Mufioz, A Ros Barcel6* Department of Plant Biology (Plant Physiology). University of Murcia, Campus of Espinardo. E-30071 Mmz'ia. Spain (Received 11 March 1991; accepted 5 December 1991)
Summary m The oxidation of hydroquinone by two peroxidasc (EC 1.1 I. !.7) fractions obtained from the cells and spent medium of cell cultures of grapevine (Vitis vin[ft,racv Monastrell) has been studied, and their comparative efficacy (kJKM ratio) studied in both the H,,O.,-consuming and hydroquinone-consuming reactions. While the efficacy in the H,,O2-consuming reaction is practically identical for both enzyme fractions, the cellular peroxidase has five-fold more efficacy in the hydroquinone-consuming reaction than the peroxidase located in the spent medium. Screening of cellular peroxidases capable of oxidizing hydroquinone on polyacrylamide gels, by means of a staining reaction based on the nucleophilic attack of 4-aminoantipyrine on p-benzoquinone in acidic media, reveals that all the cellular peroxidase isoenzymes are capable of oxidizing hydroquinone, probably yielding a quinone-diimine as a product of the staining reaction. Since isoperoxidases found in cellular fractions are also present in the spent medium, the values found for the different efficacies in the hydroquinone-consuming reaction must be considered as the results of the different proportions in which each peroxidase isoenzyme was found in the two fractions. The localization of a benzoquinone-generating system of high efficacy inside the plant cell, and probably located in vacuoles, is discussed with respect to the harmful role which the quinone/semiquinone pair might play in cell death, as part of the hypersensitive response expressed within the mechanism of plant disease resistance. peroxidase/hydroquinone oxidation/benzoquinone/grapevine Introduction Phenolic compounds form one of the most prominent classes of natural products in plants. They are ,~ery reactive chemically and are easily subjected to oxidation, substitution, and coupling reactions [1]. The degradation of p-hydroxybenzoic acids has been especially investigated in connection with decarboxylation and O-demethylation reactions [ 1]. Thus, the metabolism of p-hydroxybenzoic acids constitutes a wellknown route to simple phenols, the formation of hydroquinones being a good example. The hydroxylation-induced decarboxylation of p-hydroxybenzoic acids to hydroquinones is catalysed by peroxidases both in in vitro and in vivo conditions [21, using stoichiometric amounts of H:O2. If H_,O2 is present in stoichiometric amounts, hydroquinones are considered as end products of these catabolic reactions, and can be further glucosylated through glucosyltransferases to monoglucosides [3]. Arbutin, the monoglucoside of hydroquinone, is the
Abbreviations: AAE 4-aminoantipyrine; BQ, p-benzoquinone; HQ, hydroquinone; MN, 4-methoxy-o~-naphthol *Correspondence and reprints
principal active component of Arctostaphylos uvaursi, and is used as a urethral disinfectant, and a potent suppressor of the synthesis of melanins in animals 131. When H:O: is present in excess, hydroquinones are oxidized by peroxidases to the semiquinone radical, which is very unstable and rapidly transformed to paraquinones, dimers or polymers, depending on the substitution pattern. In this reaction, superoxide is generated, since the semiquinone radical intermediate has a high affinity for oxygen [4]. Superoxide radicals can further react with hydrogen peroxide, formed through the enzymatic or spontaneous dismutation of the superoxide radical, to form even more damaging species of oxygen, such as hydroxyl radicals and singlet oxygen [5]. In view of the multiple deleterious effects of semiquinones on plant cells, the oxidation of hydroquinone-type phenols by peroxidase would seem to be of great importance during aging stages 131 and, therefore, during the hypersensitive necrotic reaction of grapevines shown in response to fungal attack, as part of the mechanism of disease resistance I6]. In this report, we study the capacity of a cellular and a spent medium peroxidase fraction obtained from
144 grapevine cell cultures to oxidize hydroquinone (HQ) to p-benzoquinone (BQ). Further, a zymographic method is developed to screen grapevine peroxidase isoenzymes capable of oxidizing HQ through the coupling properties of the p-benzoquinone end-product with 4-aminoantipyrine (AAP). Finally, the possible participation of cellular peroxidases, through the oxidation of hydroquinone, in the previously described hypersensitive (necrotic) reaction in grapevines is discussed.
Materials and methods Reagents Hydroquinone was obtained from BDH Chemicals Ltd (Dorset, UK) and p-benzoquinone from Fluka Chemie AG (Buchs, Switzerland). Guaiacol and H20, were obtained from Merck (Darmstadt, Germany) and 4-methoxy-ot-naphthol from Aldrich Chemie (Steinheim, Germany). Ampholines and 4-aminoantipyrine were obtained from Sigma Chemical Co (St Louis, MO, USA). All other chemicals used in this work, including plant cell culture reagents, were the best available commercial products.
Materials Callus cultures were obtained from immature pericarp tissue of Monastrell grapes (Vitis vinifera L cv Monastreli) of approximately 5 mm diameter. The culture medium, based on that of Gamborg et a! [7], was supplemented with casein hydrolysate (250 mg/1), kinetin (0.2 ml/l), or-naphthalene acetic acid (0.1 mg/1) and Morel's vitamins [8]. Cultures were maintained in the dark and subcultured at approximately three-week intervals. Starting from friable callus subcultured for 24 months, grape cells were cultured in suspension on the above described culture medium in 100 ml flasks with orbital shaking (125 rpm). Suspension cultured cells were grown at 25°C in darkness for 15 days. Peroxidase from the spent medium of the cell culture was obtained by filtration of the cells through a Btichner funnel. Peroxidase from the cells was obtained by homogenization of the cells recovered in the filter in an Omni-mixer mechanical blade in the presence of 0.1 M Na-Mes buffer at pH 6,5 and 2.5c/~ (w/v~ sucrose, and centrifugation as already described 191. Both peroxidase fractions were dialyzed against 50 mM Tris-HCI buffer at pH 7.5 and stored at -20°C.
Kinetic assays Unless otherwise noted, the spectrophot~netric assay was performed at 25"C in a 1.0-ml assay volume, containing 7.5 mM hydroquinone, 0.5 mM H,,O.,, and 100 mM Tris-acetate buffer at pH 5.0. After mixing, the enzymatic reaction was initiated by adding 25 ~tl of enzyme. The increase in absorbance at 250 nm over a time period of ! rain was measured, the absorbance increase always being linear with respect to time. The oxidation rate was expressed as nmol of product formed min-I using a e,~, = i.9 x ilM M-I cm-I for p-benzoquinone [10l. The assay of peroxidase activity using 4-methoxy-o~-naphthol as substrate was carried out as described in [ ! I l, and the enzyme amounts tin nkat) in the assays were calculated using this substrate.
Zymographic assays Isoelectric focusing of grapevine peroxidase isoenzymes was performed on 3.5-10.0 pH gradients as described elsewhere [! 2]. Staining with 4-methoxy-ot-naphthol as subs(rate was carded out as previously described [11 ]. The reaction of peroxidase isoenzymes with hydroquinone was demonstrated using an incubation medium identical to that described above in the kinetic assay. The reaction was performed at 25°C for 30 min. After removal of the hydroquinone-containing reaction media, the staining reactian was developed by adding 50 mM of 4aminoantipyrine in 0. l M HCI. The red-wine colour of the product was seen l0 min after the initiation of the coupling reaction, and was stable for at least 0.5 h. Densitometric recording of 4-methoxy-ot-naphthol stained isoenzymes was carried out as described I! II, using a JoyceLoebi MK It scanner densitometer.
Results Characterization of the oxidation of hydroquinone by two grapevine peroxidase fractions The oxidation of hydroquinone (HQ) by peroxidases can be followed spectrophotometrically, since the pbenzoquinone (BQ) end-product has a characteristic absorption band in the region 2 3 5 - 2 6 8 [10]. Thus, oxidation of hydroquinone by grapevine peroxidases can be monitored by the increase of absorbance at 250 nm, which is accompanied by a decrease in the absorbance at 290 nm of the reaction media over time (fig I A). The appearance o f an isosbestic point at X = 268 nm in the consecutive spectra o f the reaction media for high reaction times or high e n z y m e concentrations seemed to suggest that there was a constant transformation of HQ to BQ, the two species (substrate and product) being kinetically correlated. The formation of BQ in these reaction media was strictly dependent on the addition of H202. However, a certain chemical oxidation took place in the absence of e n z y m e (fig IB). These observations, together with the inability of grapevine catechol oxidase (o-diphenol: 02 oxido~eductase, EC 1.10.3.1) to oxidize HQ [13], and the fact that it was not possible to determine laccase (pdiphenol:O2 oxidoreductase, EC 1.10.3.2) activity in grapevine protein fractions by means of the syringaldazine test [14], suggest that the oxidation o f HQ to BQ must be attributed to peroxidase activities. In order to characterize the peroxidatic oxidation of HQ by cellular and spent m e d i u m peroxidases, the oxidation rate (estimated from the increases in absorbance at 250 nm) was e x a m i n e d as a function o f the pH, and of both n202 and HQ concentration. Variations in the pH o f a 100 m M Tris-acetate buffer in the 3.0-7.0 range did not drastically modify the HQ oxidation rate by either cellular or spent m e d i u m peroxidase activities (fig 2), although an
145
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