ARTICLE IN PRESS Radiation Physics and Chemistry 78 (2009) 33–36
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Gamma irradiation effect on the enzymatic activities of horseradish and apple peroxidases M. Constantinovici a, D. Oancea b, T. Zaharescu c, a b c
S.C. Biotehnos S.A., 3-5 Gorunului Street, Otopeni, RO 075100, Romania Faculty of Chemistry, University of Bucharest, 4-12 Elisabeta Bd., Bucharest RO 030018, Romania Department of Radiation Processing, INCDIE ICPE CA, 313 Splaiul Unirii, P.O. Box 149, Bucharest 030138, Romania
a r t i c l e in fo
abstract
Article history: Received 15 June 2008 Accepted 24 July 2008
The behavior at low-dose exposure (0.033–0.4 kGy) of horseradish peroxidase (HRP) and of two different purified fractions of apple (Jonathan cultivar) peroxidases (named APR1S and APR2S) was studied. The HRP solutions were added with either 0.32 M fructose or glucose in order to study their effect on enzymes activity response under g (137Cs, dose rate 0.4 kGy/h) irradiation. The obtained results showed similar behavior between HRP-sugar-added solution and apple fraction with higher oligosaccharides content (APR2S) undergoing low-dose treatment. The same pattern was observed between unglycosylated HRP and APR1S with lower oligosaccharides content. These similarities gave us the possibility to conclude that the presence of oligosaccharides, in more or less quantities, influences in the same way the peroxidases activity, from different plant species, exposed to g irradiation. & 2008 Published by Elsevier Ltd.
Keywords: Peroxidase activity Gamma irradiation Inactivation
1. Introduction Peroxidases (EC 1.11.1.7) are the enzymes responsible for the occurrence of most of the degradation processes in fresh and processed vegetal food: disflavoring, bleaching/browning, and detexturing. These enzymes are involved in a great number of oxidative reactions, such as degradation of chlorophyll, phenols oxidation, biosynthesis of lignin, and many of these transmutations are related to the flavor, color, texture, and nutritional qualities of foods (Robinson and Eskin, 1991). Peroxidation is a prominent factor in apple senescence and also in fruit juice production, where some peel is also included in formulations, and this contributes to the increase in the activity of the peroxidase. The quality control imposes the assessment of peroxidase activity during fruit and vegetables processing and storage. Their remarkable heat stability and regeneration obliged food processors to apply too severe blanching conditions that affect the quality of the canned or frozen product through deterioration of color, consistency, flavor, as well as loss of valuable nutrients (Serrano-Martı´nez et al., 2008). The stability of peroxidase is influenced by their form (soluble or insoluble), as well as by the presence in that medium of some additives as sugars, which seem to have a destabilizing effect. This behavior depends on their reduction capacity, when sugars act on the conformation of the active site during the applied physico-
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chemical inactivation procedure (Chang et al., 1988). On the other hand, peroxidases are glycoproteins containing 18–37% carbohydrates, with a carbohydrate composition consisting of galactose, arabinose, xylose, fucose, mannose, mannosamine, and galactosamine depending upon the assayed isozyme and the species involved (Thongsook and Barrett, 2005). Their complex structure makes advanced peroxidases purification difficult and, thus, most inactivation experiments are done with partially purified extracts that exhibit characteristic enzymatic activity (Serrano-Martı´nez et al., 2008; Rudra Shalini et al., 2008). Since 1988, irradiation processing is a technology recommended by World Health Organization as an efficient method for the reduction of the contamination with microorganisms and for enhancing the stability of food products (Ehlermann, 2002). Actually, irradiation inactivation with high-energy radiation is an alternative method often used in commercial food processing, one of its purposes being enzyme inactivation, which always accompanies sterilization. The key parameter of irradiation is the absorbed dose, which determines sterilization (410 kGy), pasteurization (1–10 kGy), or disinfection effects (o1 kGy) (Kim et al., 2008). The primary products of water radiolysis, especially solvated electrons, are responsible for reducing the effect observed on enzymes and other biological compounds. The mechanism of water radiolysis and the activities of the main intermediates is presented in detail by Getoff (2007). The irradiation inactivation takes place through a mechanism based on the accumulation of conformational modifications, which are retrieved as plate zones in the curves of enzymatic activity dependence on irradiation dose. It was reported that the use of
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different types of media for peroxidase inactivation together with the study of substrate specificity during radiolysis allow a detailed investigation of the enzyme catalytic mechanism (Orlova et al., 1998). The aim of this study was a comparison between the properties of horseradish peroxidase (HRP) and some crude extracts of soluble apple peroxidase, which usually undergo inactivation during the processing of apple-made products. Thus, the variation in the catalytic activities of HRP and apple peroxidase during g (137Cs, dose rate 0.4 kGy/h) irradiation in different medium compositions (with or without added sugars) is presented in this paper. Addition of sugars (fructose or glucose) in the assayed medium was done in relation to the presence of usual sugars in processed vegetal products and their direct effect on different plant species peroxidases undergoing irradiation inactivation.
2. Experimental 2.1. Materials HRP was provided by Merck (170 U/mg solid) and all the other reagents were of Sigma analytical grade. The apples used for the extraction of soluble peroxidase forms were Jonathan cultivar and they had commercial provenance. 2.2. Extraction The extraction of soluble-type peroxidase from apple pulp was performed according to the method described by Valderrama and Clemente (2004). Thus, apple pulp is homogenized in an Ultraturax homogenizor with cold sodium phosphate buffer 100 mM, pH 6.5 (1:1 w/v). After homogenization, the sample is filtered through double-layered cotton tissue and the filtrate is centrifuged to 17,000g for 20 min at 4 1C. The collected supernatant was the ‘‘soluble peroxidase’’ and is stored at 18 1C until further analysis. The crude enzymatic extract was concentrated by precipitation of the protein content using cold acetone (2:1 ratio of acetone/ extract). The precipitate was collected by centrifugation and then resuspended in 100 mM sodium phosphate buffer, pH 6.5, in a volume five times smaller relative to the initial volume of the extract. A filtration step using a microfilter (0.22 mm) was applied on the resuspended precipitate. The clarified enzyme extract was dialyzed overnight in sodium phosphate buffer 10 mM, pH 7.0, at 4 1C. A 16 mm column packed with gel Sephacryl S-100-HR (Sigma Aldrich) pre-equilibrated with the eluent, 100 mM sodium phosphate buffer, pH 6.0, was used. A 5 ml sample was applied to the column; the flow rate was 30 ml/h. Fractions of 5 ml were collected and assayed for POD activity and protein content. Two fractions with evident peroxidasic activity, APR1S and APR2S (experimental marks ascribed to the two isolated apple extracts, the abbreviations from APple extRact), were further characterized. The total protein content of apple-soluble peroxidase extracts was determined in conformity with the Bradford method (Bradford, 1976) and it was 0.1 mM for APR1S and 0.5 mM for APR2S. The purity of the two apple peroxidasic fractions was further analyzed using SDS-PAGE electrophoresis under reducing conditions, applied on a Mini-Protean II system (BioRad, USA). The polyacrylamide stacking gels had different composition from the separation ones, as described by Thongsook and Barrett (2005). The following molecular weight markers used for electrophoresis were provided by BioRad: myosine (200 kDa), galactosidase (116 kDa), phosphorylase b (97 kDa), serum albumin (66 kDa),
ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa), and aprotinin (6.5 kDa). The Coomassie blue staining technique was used. The method used for estimating the initial and residual enzymatic activities of HRP and apple peroxidase has been based on the spectrophotometric monitorization of peroxidasic reaction of 0.7 mM guaiacol in the presence of 1 mM H2O2 and 0.2 M sodium phosphate buffer, pH 6 (Bhamidipati and Singh, 1996). The increasing rate of absorbance at 470 nm due to the formation of brown products of guaiacol oxidation was measured with a JASCO W32 spectrophotometer (Japan). The peroxidasic activity was determined from the slope of the linear regression plot over the first 40 s and expressed as A470 nm s 1 mg 1 enzyme. The HRP concentration ranged between 0.5 and 25 mg/mL during the experiments. 2.3. Irradiation The exposure of samples was carried out in an irradiator, GAMMATOR M-38-2 (USA), with the dose rate of 0.4 kGy/h. A part of these experiments were carried out in sugar-added environment (0.32 M glucose or fructose) in order to screen their effects on enzymatic peroxidase activity during irradiation. The APR1S and APR2S were exposed to the same experimental conditions as HRP. The HRP and apple peroxidase probes were prepared in 0.2 M sodium phosphate buffer pH 6.0. Periodically, aliquots were taken from irradiated probes and their residual activity was measured according to the above-described method. The used irradiation doses varied between 0.033 and 0.4 kGy depending on the initial enzymatic activities of the studied probes. Each experimental point was calculated from five independent replicates with an error of 75%.
3. Results and discussion The results of SDS-PAGE gel electrophoresis of APR1S and APR2S fractions of the two apple peroxidase fractions are presented below (Fig. 1). The first line presents the migration of molecular weight standards and lines 2 and 3 present the fractions of apple peroxidases. Both of them have molecular weights near ovalbumin (45 kDA) and carbonic anhydrase (31 kDa), these results being in accordance with the data literature (Valderama and Clemente, 2004; Khan and Robinson, 1993). Molecular weights of peroxidases from different sources range from 30 to 60 kDa and the differences are attributed to post-translational modifications of the polypeptide chain, including the number and composition of glycan chains present in their native structures. This characteristic of glycoproteins is similar for many plant peroxidases, having oligosaccharide chains linked to asparagine residue. The carbohydrate moieties of horseradish (HRP), turnip, Japanese radish, and oil palm leaf peroxidases contain ca. 18%, 12–18%, 20%, till 37% carbohydrate bound to the protein moiety, respectively (Deepa and Arumughan, 2002; Kim and Kim, 1996), but with any particular glycan function. It can be seen from the above electrophoregram that the APR2S fraction has a higher molecular weight given by more oligosaccharides residues present in its composition than in the APR1S fraction. This fraction, which was eluted later during the gel chromatography purification procedure, probably has less sugar chains and, thus, lower molecular weight than the APR2S. The residual enzymatic activities (ratio of final/initial peroxidase activity, expressed as REA, %) were plotted against the irradiation dose for each of the studied probes (Figs. 2 and 3 for APR1s and APR2S, respectively).
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35
120 aprotinin (6.5kDa)
100
REA (%)
lysozyme (14.4 kDa) trypsion inhibitor (21.5 kDa) carbonic anhydrase (31 kDa) ovalbumin (45 kDa)
80 60 40
serum albumin (66 kDa)
20 phosphorylase b (97 kDa) galactosidase (116 kDa)
0 0.0
myosine (200 kDa)
1
2
3
0.1
0.2 Dose (kGy)
0.3
0.4
Fig. 3. REA of APR2S and of HRP 0.5 mg/ml+glucose or fructose 0.32 M at various irradiation doses: (&) APR2S 0.1 mg/ml; (J) HRP 0.5 mg/ml+glucose 0.32 M; (n) HRP 0.5 mg/ml+fructose 0.32 M.
Fig. 1. SDS-PAGE electrophoresis of apple peroxidase fractions compared with molecular weight standards: (1) molecular weight standards; (2) APR2S; (3) APR1S.
tional states, which coexist in chemical equilibrium in comparison with unglycosylated peroxidases (HRP and APR1S), where their smaller number helps them to accept more rapidly the inactivation in increasing the irradiation dose. The explanation of these oscillations is based on the hypothesis of saturation effect appearing at the conformational modification of an enzyme structure, which is proposed by Dubrovsky et al. (2003). In fact, the results were the consequence of the radiolysis effects on water according to the comprehensive mechanism reported by Wood and Pikaev (1993) and on peroxidases whose molecules can be scissored at their weakest sites.
150
REA (%)
125 100 75 50
4. Conclusion
25
The irradiation inactivation mechanism takes place most probably through enzymic structure disorganization following the transitional conformational states appearing during increasing irradiation dose. The energy transfer from incidental radiation imposes the stochastic degradation of some amino acid residues, which, finally, leads to enzymic architecture degradation. The irradiation with controlled doses of peroxidase is a viable method for their inactivation in fresh and processed vegetal foods, taking into account the optimization of the applied irradiation dose and the medium/food composition.
0 0.0
0.1
0.2 Dose (kGy)
0.3
0.4
Fig. 2. Dose dependencies of residual enzymatic activity (REA) of horseradish and apple peroxidases: (B) HRP 25 mg/ml; (&) HRP 5 mg/ml; ( ) HRP 2.5 mg/ml; (n) HRP 1 mg/ml; (+) HRP 0.5 mg/ml; (J) APR1S 0.5 mg/ml.
It is obvious that HRP and APR1S present the same mild but continuous pattern of inactivation process without any rude variations of enzymatic activity, suggesting the inactivation– activation phenomena during irradiation (Fig. 2). The plots of HRP in sugar environment (0.32 M glucose or fructose) and APR2S present rude variations of enzymatic activity, which appear with increasing irradiation dose (Fig. 3). These similarities between the two enzymatic probes could be due to the fact that crude extracted peroxidases from vegetal sources present some oligosaccharide chains that stabilize its conformation. These sugar chains probably exist in apple enzymatic probe APR2S, but they were eliminated during a more advanced purification of APR1S, explaining the similar action of APR2S and HRP with sugars. The HRP-amplified structures with added sugars, together with APR2S, generate probably a much higher number of conforma-
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