Purification and Characterization of Peroxidase from Turkish Black Radish (Raphanus Sativus L.)

Full Length Research Paper

Purification and characterization of peroxidase from Turkish black radish (Raphanus sativus L.)

Melda Şişecioğlu1, İlhami Gülçin1,2*, Murat Çankaya3, Ali Atasever4, M. Hilal Şehitoğlu1, Habibe Budak Kaya1 and Hasan Özdemir1

1Department of Chemistry, Faculty of Sciences, Atatürk University, 25240-Erzurum, Turkey. 2School of Health Services, Ibrahim Çeçen University, TR-4100-Agri-Turkey. 3Department of Biology, Faculty of Sciences, Erzincan University, 24100 Erzincan, Turkey. 4Department of Food Sciences, Ispir H. Polat Vocational School, Atatürk University, 25900 Ispir, Erzurum, Turkey.

Accepted 2 June, 2010

Peroxidases (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) are part of a large group of enzymes associated with cell wall biosynthesis, response to injury, disease, resistance and wound repair. They catalyze the oxidation of various electron donor substrates such as phenols and aromatic amines in the presence of hydrogen peroxide. In the present study, peroxidase, a primer antioxidant enzyme, was purified 9.7 fold from Turkish black radish (Raphanus sativus L.) in a yield of 2% by ammonium sulphate precipitation, dialysis and CM-Sephadex ion exchange chromatography. To check the enzyme purity, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed and a single band was observed. The substrate specificity of peroxidase was investigated using guaiacol (2-methoxyphenol). Optimum pH, optimum temperature, optimum ionic strength, stable pH, stable temperature and thermal inactivation conditions were determined for guaiacol / H2O2 substrate pattern. These kinetic properties were found to be 6.0, 30°C, 1.0 M, 9.0, and 60°C, respectively. The molecular weight (Mw) of the enzyme was found to be 66 kDa by sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS - PAGE) method. Native polyacrylamide gel electrophoresis (Native-PAGE) was performed for isoenzyme determination and a single band was observed. Km and Vmax values were calculated from Lineweaver-Burk graphs.

Key words: Turkish black radish, Raphanus sativus, peroxidase, enzyme purification, enzyme characterization.

INTRODUCTION

Peroxidase (POD) is a heme protein, which is a member suberization (Bernards et al., 1999), auxin oxidation of oxidoreductases [E.C.1.11.1.7] and catalyses the (Gazaryan and Lagrimini, 1996), senescence (Santos et oxidation of a wide variety of organic and inorganic al., 2001), organogenesis (Lee et al., 2002) phenol substrates using hydrogen peroxide as the electron oxidation (Lagrimini, 1991), cross-linking of cell wall acceptor (Banci, 1997; Yemenicioğlu et al., 1998). proteins (Schnabelrauch et al., 1996), salt stress Peroxidases are widely distributed in living organisms tolerance (Hiraga et al., 2001) and protection of tissue including microorganisms, plants and animals. POD is from damage and infection by pathogenic microorga- mainly located in the cell wall (Chen et al., 2002) and it is nisms (Sakharov et al., 2000; Gülçin and Yildirim, 2005; one of the key enzymes controlling plant growth and Sat, 2008). development. It takes place in various cellular processes POD and catalase are two major systems for the including construction, rigidification and eventual enzymatic removal of H2O2 and peroxidative damage of lignifications of cell walls (Quiroga et al., 2000), cell walls is controlled by the potency of antioxidative peroxidase enzyme system (Velikova et al., 2000). However, the role that peroxidase plays in metabolism is not clear because of the large number of reactions it *Corresponding author. E-mails: [email protected], igulcin@ catalyzes and the considerable number of isoenzymic yahoo.com. Tel: +90 442 2314444. Fax: +90 442 2360948. species (Kim and Lee, 2005). Therefore, POD is also

1188 J. Med. Plant. Res.

widely used for clinical diagnosis and microanalytical MATERIALS AND METHODS immunoassays because of its high sensitivity. New applications for POD have been suggested in the Chemicals and apparatus medicinal, chemical and food industries (Kwak et al., Guaiacol, CM-Sephadex A-50 and Coommassie Brilliant Blue R- 1996). Other applications include synthesis of various 250 were obtained from Sigma Aldrich Chemie GmbH. Co. MBP- aromatic compounds and removal of peroxide from truncated-β-galactosidase, MBP-CBD, aldolase and triosepho- foodstuff and industrial wastes (Aruna and Lali, 2001). sphate isomerase were purchased from New England Concerning the physiological role of peroxidases, it has (http://www.neb.com/nebecomm/products/productP7709.asp). A refrigerator (Sanyo, Ultra low, -86°C) was used for protection of been shown that the enzyme participates in the formation Turkish black radish homogenate. The changes in absorbance at of lignins in the secondary cell walls during normal growth 470 nm were recorded using a double beam UV-VIS (Pedreno et al., 1995) and in the formation of phenolic spectrophotometer (CHEBIOS s.r.l.). A pH-meter (SCHOTT polymers such as lignins, suberins, etc. when plants are CG840) was used for adjusting the acidity. infected or wounded (Dixon and Palva, 1995; Köksal and Gülçin, 2008). It was reported that peroxidase had been Plant materials used for biotransformation of organic molecules (Dordick et al., 1987; Adam et al., 1999; Gülçin and Yildirim, Fresh Turkish black radish (R. sativus L.) was cultivated in 2005). Because of its broader catalytic activity, a wide Hasankale region of Erzurum province, Turkey. It was washed, range of chemicals can be modified using POD. Also, it drained, packed in polyethylene bags and stored at -83°C until can be used for the applications such as synthesis of used. various aromatic compounds, removal of phenolics from waste waters and the removal of peroxides from Preparation of Turkish black radish (Raphanus sativus L.) foodstuffs, beverages and industrial wastes (Torres et al., homogenate 1997). POD is also related to quality of plant commo- dities, particularly the flavor, in both raw and processed The homogenate preparation procedures for POD were adapted foods. POD activity is also correlated to fruit ripening as from the methods described by Sakharov et al. (2002). For this purpose, 20 g Turkish black radish were taken from frozen storage shown in a number of cases and it is also involved in (-83°C) and ground in a mortar in the presence of liquid Nitrogen. enzymatic browning, either or together with polyphenol This powder then mixed with 50 ml of phosphate buffer (pH: 7.0, oxidase activity. A more precise understanding of the 0.3 M) and subsequently the slurry was centrifuged at 15.000 x g implication of POD in these mechanisms is an essential for 60 min at 4°C (Gülçin, 2002). The buffer composition was as step towards a more efficient control of these undesirable follows: 0.1 M phosphate buffer; PVP (0.05%, pH: 7.0). The pellet reactions, particularly in heat-processed products, which was discarded. frequently contain residual peroxidase activity (Cardinali et al., 2007; Köksal and Gülçin, 2008). Ammonium sulphate fractionation and dialysis Although, peroxidases are widely distributed in the plant kingdom, the major source of commercially Ammonium sulphate precipitation was carried out in homogenate available peroxidase is roots of horseradish. On the other on an ice bath for 0 - 10, 10 - 20, 20 - 30, 30 - 40, 40 - 50, 50 - 60, hand, availability of peroxidases with different specificity 60 - 70 and 70 - 80% intervals. Ammonium sulphate was slowly added to the homogenate stirred until complete dissolution. Then would promote the development of new analytical the mixture was centrifuged at 15.000 x g for 60 min and the methods and potential industrial processes (Köksal and precipitate was dissolved in 2 ml of phosphate buffer (pH: 7.0, 0.3 Gülçin, 2008). Turkish black radish (Raphanus sativus L.) M). The concentrated sample with maximum specific activity was is cultivated in all regions of Turkey. Turkish black radish selected and dialyzed in a dialysis tube for 12 h against 1 L above peroxidase activity was evaluated after high-pressure buffer at 4°C for further use (Köksal and Gülçin, 2008). treatment with carbon dioxide (Fricks et al., 2006). Moreover, regulation of the activity of the Korean radish Preparation of CM-Sephadex A-50 ion exchange cationic peroxidase promoter during dedifferentiation and chromatography material redifferentiation were reported (Kim et al., 2004). Ascorbate peroxidase was also purified and Briefly, 3.5 g dried CM-Sephadex A-50 (Sigma) was dissolved in characterized from roots of Japanese radish (Ohya et al., 100 ml distilled water and incubated in a 90°C water bath for 5 h. Following cooling to the room temperature, this slurry was mixed 1997). In addition, a plant peroxidase localized in the root with 100 ml NaOH (0.5 M) and was allowed to stand for 1 h. tissues of Turkish black radish was purified partially by Afterwards, the supernatant was decanted and the exchanger was precipitation using a reversibly soluble/insoluble ion- washed with distilled water until the effluent is at neutral pH. Then, exchange polymer system of CM-cellulose, calcium and the exchanger was stirred in 100 ml 0.5 M HCl and allowed to stand polyethylene glycol (Aruna and Lali, 2001). The objective for an additional 1 h. Subsequently, the exchanger was washed of this study was the purification and characterization of with distilled water until the pH of the effluent was 7.0. Finally, the exchanger was suspended in 0.01 M phosphate buffer (pH: 6.5) peroxidase from Turkish black radish grown in the then packed in a column (3 x 30 cm), washed and equilibrated with Anatolia. the same buffer. The flow rates for washing and equilibration were

Sisecioglu et al. 1189

SDS-PAGE electrophoresis 120 1 Discontinuous polyacrylamide gel electrophoresis was performed

100 undernm) denaturing conditions after POD purification according to the 0.8 method of Laemmli (1970) and described previously (Beydemir et al., 2005; Şenturk et al., 2008; Gülçin et al., 2008). 40 mg samples 80 280 0.6 were applied to the electrophoresis medium. Gel was stained overnight in 0.1% Coomassie Brilliant Blue R-250 in 50% methanol 60 and 10% acetic acid, then distained by frequently changing the 0.4 same solvent, without dye (Beydemir et al., 2003; Beydemir and 40 Gülçin, 2004; Ekinci et al., 2007; Şişecioğlu, 2008; Ozturk Sarikaya et al., 2010). 20 0.2

( Absorbance Molecular weight determination 0 0

The molecular weight of peroxidase from Turkish black radish was POD Activity (%) Activity POD 1 5 9 1317212529333741454953576165 estimated by SDS - polyacrylamide gel electrophoresis (Weber et al., 1972). The protein bands were stained with Coomassie Brilliant Blue R-250. The standard proteins used for SDS-PAGE were MBP- Figure 1. CM-Sephadex A50 ion exchange chromatography of truncated-β-galactosidase (83 kDa), MBP-CBD (62 kDa), Aldolase POD from Turkish black radish (FractionRaphanus sativusnumber L.): Elution (47.5 kDa) and Triosephosphate isomerase (32.5 kDa) (MBP: profile of unbound fraction from CM-Sephadex A-50 obtained in Maltose binding protein, CBD: Chitin binding domain). 0.1 M sodium phosphate buffer (pH: 7.0) as 3 ml fractions. The absorbance of protein at 280 nm is presented on the left hand y- axis whereas the POD activity of individual fraction is presented Peroxidase activity assay on the right hand y-axis. The POD activity in the Turkish black radish sample was measured using guaiacol substrate. When studying substrate specificity of POD, the activity was measured under optimal conditions for each adjusted to 40 ml h-1 by peristaltic pump (Robyt and White, 1987). substrate. Temperature was controlled using a circulating water bath with a heater/cooler (Grant LTD 6G -20 to 100°C, England). Initial rates of free radical formation for substrates were monitored at maximum wavelength of each substrate. The changes in Purification of peroxidase by CM-Sephadex A-50 ion exchange absorbance were read for 3 min using a double beam UV-VIS chromatography spectrophotometer (CHEBIOS s.r.l.) at the 470 nm using by guaiacol substrate. Briefly, an aliquot of enzyme sample (10 µl) was Dialyzed Turkish black radish sample was loaded onto previously added to a mixture of 1 ml 22.5 mM H2O2, 45 mM guaiacol (1 ml), equilibrated CM-Sephadex A-50 column and the gel was washed and final volume of this mixture was adjusted to 3 ml by addition of with 1 L of phosphate buffer (pH: 7.0, 0.1 M). Then, dialyzed POD phosphate buffer (pH: 6.0, 0.1 M). The changes in the absorbance enzyme sample (14 ml) was loaded onto the CM-Sephadex A-50 at above wavelength were monitored for 3 min at 20°C. One unit of ion exchange column. Bound proteins were eluted with a gradient enzyme activity is defined as 0.01 change of absorbance at each of (250 ml) 0 - 1 M NaCl in 10 mM phosphate buffer pH: 7.0 at 15 wavelength, depending on the substrates (Fujita et al., 1997; Gülçin ml h-1 flow rate. Eluates were collected as 3 ml fractions (Figure 1) et al., 2005; Gülçin et al., 2009). and each activity and absorbance were separately measured at 470 nm and 280 nm, respectively (Fujita et al., 1997). Active fractions were pooled and kept at + 4°C until use. Qualitative and quantitative protein determination

Qualitative and quantitative protein determination was described previously (Çoban et al., 2007; 2008) Qualitative protein Native PAGE electrophoresis determination was done at 280 nm on the eluates obtained and POD activity was measured in the eluates showing absorbance at Native polyacrylamide slab gel electrophoresis (PAGE) was 280 nm. Quantitative protein determination was achieved by performed according to Laemmli’s procedure (1970) under native absorbance measurements at 595 nm according to Bradford’s conditions (that is, without sodium dodecyl sulfate) for separating method (1976), with bovine serum albumin as standard (Gülçin et POD isoenzymes (Gülçin et al., 2005). The experiment was al., 2004; Şentürk et al., 2009; Çoban et al., 2009; Innocenti et al., conducted in a cold room at + 4°C with the electrode buffer 2010). Tris/Glycine (pH: 8.3) using 3% stacking gel and 10% separating gels. 20 µl enzyme samples were loaded on to each space of the stacking gel. Initially, an electric current of 80 V was applied until Kinetic Studies the bromophenol dye extended into the separating gel and then increased to 150 V for 5 - 6 h until the tracking dye migrated to 1 Optimum pH profile cm from the bottom. After running, gels were incubated in 4.5 Mm guaiacol and 22.5 mM H2O2 in 100 mM phosphate buffer (pH: 7.0) The optimum pH value for POD activity was found by assaying at 37°C until appearance of the enzyme bands and enzyme activity at different pH levels. The assay was carried out in thenphotographed (Köksal and Gülçin, 2008). the presence of buffers with different pH such as (0.1 M sodium 1190 J. Med. Plant. Res.

Table 1. Levels of purification of Turkish black radish (R. sativus L.) peroxidase obtained after the application of different purification steps leading to the improvement in the activity of enzyme.

Total Enzyme Total enzyme Total Specific Protein Yield Purification Purification steps volume activity activity -1 protein activity -1 (mg ml ) (%) fold (ml) (EU ml ) (EU ml-1) (mg) (EU mg-1) Homogenate 120 54000 6480000 0.0909 9.09 24158.4 100 1

(NH4)2SO4 precipitation 13 844000 10972000 0.2121 2.5452 32795.9 28 1.358 Dialysis 14 806000 11284000 0.1393 1.72 42670.5 19 1.766 CM - Sephadex ion exchange 38 24400 927200 0.0097 0.194 235051.6 2 9.7 chromatography

acetate, pH: 3.0 - 4.5; 0.1 M sodium phosphate, pH: 4.5 - 7.5; 0.1 M radish. Km and Vmax values were determined for guaiacol/H2O2 Tris-HCl, pH: 8.0 - 9.0) pH: 3.0 - 9.0 separately, in an assay mixture substrate pair. For this, the enzyme activity was measured at five (Sakharov et al., 2002; Köksal and Gülçin, 2008). different concentrations of guaiacol at constant H2O2 concentration. In addition, this measurement was performed at a constant concentration of guaiacol whilst five different H2O2 concentrations pH stability profile were used. Km and Vmax values were calculated from the plot of 1/V versus 1/[S] by the method of Lineweaver and Burk (1934). The crude POD from Turkish black radish showed maximum activity at pH ranging from 4.0 to 7.5. The enzyme was stable over a wide range of pH from 4.0 to 9.0 exhibiting highest stability at pH: 7.0 in Statistical analysis the phosphate buffer.

The results regarding Km and Vmax values are given as ± S.D. of four parallel measurements. Analysis of variance was performed by The effect of temperature t-test.

POD activity was determined at various temperatures controlled by a circulatory water bath. The POD activity at a definite temperature was determined spectrophotometrically by addition of enzyme to RESULTS the mixture as rapidly as possible. For determining optimum temperature of the enzyme, guaiacol/H2O2 substrate patterns were Ammonium sulfate fractionation was done by using the used. The activity was measured at different temperatures in the finely ground ammonium sulfate. The fraction obtained in range from 0 to 80°C. In order to determine the thermal stability of the POD, we used above mentioned substrates. The purified 20 - 60% interval showed the maximum activity. This enzyme was assayed at 30, 40, 50, 60, 70 and 80°C. For the study, primary purification step resulted in about 1.4 - fold 1 ml of enzyme solution in a test tube was incubated at the required purification of POD from the crude extract (Table 1). The temperature for fixed time intervals (10, 20, 30, 40, 50 and 60 min). dialyzed enzyme extract was applied to a CM-Sephadex At the end, the enzyme was cooled to room temperature in an ice A 50 ion exchange column and bound proteins were bath. Under optimal conditions, 0.1 ml heated enzyme extract was mixed with substrates and buffer, and residual POD activity was eluted with a linear gradient of 0 - 1 M NaCl in 10 mM determined spectrophotometrically. The percentage residual POD phosphate buffer (pH: 7.0). POD purified from Turkish activity was calculated by comparison with unheated enzyme black radish exhibited only one band on SDS-PAGE (Köksal and Gülçin, 2008). electrophoresis, as shown in lane b of Figure 2. Also, native polyacrylamide slab gel electrophoresis (N-PAGE) was performed at +4°C under native conditions without Effect of ionic strength sodium dodecyl sulphate for separating POD enzyme The effect of ionic strength on the enzyme was checked for each under natural condition. As can be seen in lines c and d substrate using different concentrations of buffers (0.1 - 3 M). As of Figure 2, one isoenzyme was found. In addition, as shown in Table 2, POD activity varied slightly with the buffer can be seen in Figure 3, the molecular weight (Mw) of concentration ranging from 0.5 to 3.0 M. It was observed that POD POD of purified peroxidase was calculated as 66 kDa activity was maximal at 0.1 M of buffer concentration. from SDS-PAGE. The electrophoretic pattern was shown

in Figure 2 and column a. Substrate specificity The optimum pH was found by assaying enzyme activity at different pH levels. The enzyme activity was Under optimal conditions, the efficiency of catalytic oxidation of measured in different buffers: 0.2 M phosphates buffer guaiacol by hydrogen peroxide in the presence of POD was (pH: 5.0 - 7.5), 0.1 M Tris-HCl buffer (pH: 7.5 - 9.0), evaluated. Km and Vmax values were calculated for peroxidase reactions with each of the five substrates, using the Lineweaver- (Figure 4). It was found that the enzyme had the highest Burk transformation of the Michaelis-Menten equation. Table 2 activity in 0.1 M phosphate buffer at pH: 6.0 (Table 2). As shows the kinetic constants of POD purified from Turkish black shown in Table 2, the optimal pH value of POD was Sisecioglu et al. 1191

Table 2. Optimum pH, optimum temperature, optimum ionic strength, stable pH, thermal stabilization and substrate specificity of peroxidase from Turkish black radish (R. sativus L.)

Kinetic properties Guaiacol Optimum pH 6.0 Optimum temperature ( C) 30 Optimum ionic strength (M) 1.0 Thermal stabilization ( C) 60 Stable pH 9.0

Km (mM) 0.036 ± 0.08 -1 -1 Vmax (EU ml min ) 38728.17 ± 1183.24

determined as 6.0 at 0.1 M sodium phosphate buffer. standard proteins were used for the standard graph: To determine the stabile pH, the peroxidase enzyme MBP-truncated-β-galactosidase (83 kDa), MBP-CBD (62 activity was followed in three different buffers in a pH kDa), aldolase (47.5 kDa), triosephosphate isomerase range of 4.5 to 9.0 with 0.5 intervals, for 8 days. As can (32.5 kDa). (MBP: maltose binding protein, CBD: chitin be seen in Figure 5 and Table 2; POD was more stable at binding domain, Figure 2 and column a). As can be seen pH ranged 5 to 9 in 0.1 M phosphate and 0.1 M Tris/HCl in Figure 3, A Log MW-Rf volume standard graph was buffer at the end of this incubation period. The optimum obtained. Molecular weight of POD was calculated by temperature of peroxidase enzyme was determined to be using the following formula obtained in the standard 45°C for guaiacol (Figure 6). The enzyme activity was too graph: low at 80°C; hence this temperature was not studied. The effect of ionic strength on the enzyme was studied R -1.437 x Log 2.861 R 2 0.996 for each substrate using different concentrations of f MW buffers (0.1 - 3.0 M). Figure 7 and Table 2 showed that the POD activity was maximum at 1 M and varied slightly Then, Rf value of purified peroxidase was inserted into with the buffer concentration ranging from 0.5 to 3.0 M. In above equation (Beydemir et al., 2003). Thus, Mw of general, POD activity was maximal in 1 M of buffer purified peroxidase was calculated as 66 kDa. SDS - PAGE photograph was shown in Figure 3b. This value is concentration. Km and Vmax values were calculated from very different from other results. Scialabba et al. (2002) Lineweaver-Burk graphs. The enzyme had Km values of 0.036 ± 0.08 and 0.0084 ± 0.003 mM for guaiacol and showed four peroxidase isozymes, expressed in maturing radish seed, with molecular weight of 98, 52.5, 32.8 and H2O2 substrates, respectively. On the other hand, the 29.5 kDa. Also, it was known that Korean radish enzyme had Vmax values of 38728.17 ± 1183.24 and 35122.23 ± 1729.12 EU ml-1 min-1 for above substrates, isoperoxidases had molecular weight between 31 and 50 respectively (Table 2). kDa (Lee and Kim, 1994). Similarly, Köksal and Gülçin found that POD purified from fresh cauliflower (Brassica oleracea L.) buds had a molecular weight of 44 kDa by DISCUSSION gel filtration chromatography method (2008). On the other hand, in previous papers from our laboratory, Gülçin and The POD from Turkish black radish extract was purified Yildirim purified and characterized peroxidase from B. in a few steps, including ammonium sulphate oleracea which had a molecular weight of 95 kDa (2005). fractionation, dialysis and CM-Sephadex ion-exchange It was found that POD purified from fresh cauliflower (B. chromatography (Table 1). For this, firstly, the crude oleracea L.) had optimum pH at pH 6.0 when guaiacol enzyme extract was concentrated by progressive and hydrogen peroxide were used as substrates The fractionation by ammonium sulphate precipitation with 0 - optimum pH of peroxidase activity of cationic peroxidase 20, 20 - 40, 40 - 60, 60 - 80 and 80 - 100% intervals. Cs in R. sativus for o-dianisidine oxidation was observed Therefore, this saturation range was used in all of the at pH 7.0 when o-dianisidine and hydrogen peroxide extraction processes. Following ammonium sulphate were used as substrates (Kim and Lee, 2005). It was precipitation, the enzyme-containing precipitate was known that optimum pH of an enzyme varies according to dissolved in 12.4 ml of phosphate buffer (pH: 7.0, 0.3 M) substrate types. For example, the optimal pH of POD and then dialyzed against phosphate buffer (pH: 7.0, 1 L, from cauliflower (B. oleracea L.) buds was determined to 0.3 M) for 12 h. be 5.0 for the guaiacol substrate. This value was found to The molecular weight of POD was determined by SDS- be 4.0 for both ABTS and catechol substrates. For PAGE according to Laemmli’s method (1970). Four pyrogallol and 4-methyl catechol substrates, the optimum

1192 J. Med. Plant. Res.

100 80 60 40 Acetate buffer 20 Phosphate buffer Tris-HCl buffer 0

POD ActivityPOD (%) 2 4 6 8

Figure 4. Optimum pH study for POD from Turkish black radish (R. sativus L.). Figure 2. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (N-PAGE) zymogram of peroxidase from Turkish black radish (R. sativus L.). Column a: Standard proteins (MBP- Talaz et al., 2010). To determine the optimum tempera- truncated-β-galactosidase (83 kDa), MBP-CBD (62 kDa), aldolase ture of the enzyme, POD activity was measured at (47.5 kDa), triosephosphate isomerase (32.5 kDa). Column b: different temperatures in the range from 5 to 80°C for 5 purified peroxidase from Turkish black radish (R. sativus L.) by CM- min under optimal pH and buffer concentration for each Sephadex A50 ion exchange chromatography. Column c: Peroxidase obtained from Turkish black radish (R. sativus L.) and substrate. The effect of temperature on POD activity was dyed by guaiacol. Column d: Peroxidase obtained from Turkish tested by heating the standard reaction solutions (buffer black radish (R. sativus L.) and dyed by ABTS. and substrates) to the appropriate temperatures before introduction of the enzyme. The desired temperatures were provided by using a heater/cooler controller attached to the cell-holder of the spectrophotometer. 0.8 Once temperature equilibrium was reached, enzyme was added and the reaction was followed spectrophoto- 0.6 metrically at constant temperature at given time intervals. The results shoved that POD had no activity above 80°C. 0.4 POD purified from Turkish black radish had optimum Rf temperature at 30°C. Therefore, as mentioned above, optimum temperature of enzymes varies depending on 0.2 substrate types. For instance, the optimum temperature of peroxidase enzyme from cauliflower (B. oleracea L.) 0 buds was determined to be 25°C for pyrogallol, 30°C for 1.5 1.6 1.7 1.8 1.9 guaiacol and ABTS substrates, 45°C for 4-methyl catechol and 50°C for catechol (Köksal and Gülçin, Log Mw 2008). Temperature effects on the POD were carried out by Figure 3. Standard Log MW-Rf graph of POD purified from Turkish measuring the residual activity after incubating 1 ml of the black radish (R. sativus L.) using SDS-PAGE [MBP-truncated-β- enzyme at different temperatures in a water bath in the galactosidase (83 kDa), MBP-CBD (62 kDa), aldolase (47.5 kDa), triosephosphate isomerase (32.5 kDa), (MBP: Maltose binding range from 10 to 60°C under optimal pH and buffer protein, CBD: Chitin binding domain). concentration for each substrate (Figure 8). For the study, 1 ml of POD enzyme solution in a test tube was incubated at the required temperature for fixed time intervals (10, 20, 30, 40, 50 and 60 min). At the end of pH was found to be 7.5. In this study, ABTS was used as the required time interval, the enzyme was cooled to substrate for Turkish black radish POD, however, this room temperature in an ice bath. Under optimal chemical was frequently used as source of ABTS+ for conditions, 0.10 ml heated enzyme extract was mixed ABTS radical scavenging (Köksal and Gülçin, 2008; with substrates and buffer and residual POD activity was Gülçin et al., 2006; 2007; 2008; 2009; 2010; Ak and determined spectrophotometrically. The percentage Gülçin, 2008; Gülçin, 2006; 2007; 2008; 2009; 2010; residual POD activity was calculated by comparison with

100 3 3.5 Sisecioglu et al. 1193 4 85 4.5 100 5 3 70 3.5 4 10085 100 4.53 53.5 POD POD Activity (%) 55 4 80 85 70 4.5 40 5 60 70 0 2 4 6 8 POD POD Activity (%) 55 40 20

POD Activity (%) 4055 0 2 4 6 8 0 10040 5.5 0 20 40 60 80

0 2 4 6 8 ActivityPOD (%) 6 6.5 7 o 80 Temperature ( C) 100 7.5 5.5 6 Figure 6. The effect of temperature on the peroxidase activity 10060 6.5 from Turkish black radish (R. sativus L.). 80 5.57 67.5 6.5 POD Activity (%) 4080 7 60 7.5 100 2060 80 POD Activity (%) 40 0 2 4 6 8 60

POD Activity (%) 40 20 40

0 2 4 6 8 20 20 0 100 0 2 4 6 8 8 8.5 0 1 2 3 9 Acticvity (%) POD

10080 [Na 2HPO4] (M) 8 8.5 100 9 Figure 7. The effect of ionic strength on the peroxidase 8 8.5 activity from Turkish black radish (R. sativus L.). POD Activity (%) 6080 9

POD Activity (%) 4060 specificity. Km and Vmax values were determined for 0 2 4 6 8 guaiacol / H2O2 substrate pairs from Lineweaver-Burk

POD Activity (%) 60 graph. As can be see in Figure 9, the enzyme activities Time (Day) were measured at five different concentrations of 40 Figure 5. Stabile pH profile of peroxidase POD from Turkish substrates while H2O2 concentration was constant. These 0 2 4 6 8 black radish (R. sativus L.). The enzyme activity was measured measurements were also performed at a constant 40 in three different buffers: 0.2 Time M phosphates (Day) buffer (pH: 5.0 - concentration of guaiacol, whilst five different H2O2 7.5), 0.1 M0 Tris-HCl buffer2 (pH: 7.5-9.04 ). 6 8 concentrations were used. It was reported that the

Time (Day) reconstituted peroxidase from R. sativus had Km values of 1.18 mM against o-dianisidine and 1.27 mM against unheated enzyme (Köksal and Gülçin, 2008). POD, H2O2, respectively, (Kim and Lee, 2005). We found that heme -containing glycoprotein, using hydrogen peroxide enzyme had Km values of 0.036 ± 0.08 and 0.0084 ± as electron acceptor and several substrates as an 0.003 mM for guaiacol and H2O2 substrates, respectively. electron donor are involved in broad range of On the other hand Vmax values of the reconstituted physiological processes such as lignification, peroxidase Cs from R. sativus were determined to be suberization, cell wall metabolism, auxin catabolism, 0.138 EU for o-dianisidine and 0.032 EU for H2O2, biotic and abiotic stress tolerance and senescence. Thus, respectively. Peroxidase from Turkish black radish had we determined Km and Vmax values for guaiacol / H2O2 Vmax values of 38728.17 ± 1183.24 and 35122.23 ± substrate pairs in order to be able to compare substrate 1729.12 EU ml-1 min-1 for above substrates, respectively 1194 J. Med. Plant. Res.

100

80 30 40 60 50 60 40 70 80

20 POD Activity (%) Activity POD

Figure 8. The10 effect of incubation20 period30 on the peroxidase40 activity50 from Turkish60 black radish (R. sativus L.). Enzyme wasTemperature dissolved in 0.1 (o C)M phosphate buffers that have indicated pH and were incubated at +4 C. The peroxidase activity of each of incubated enzyme solutions were measured at indicated periods.

15 12

12 -1 9

9 6 6

1/Vx10-5 (EU/mL.min) 3

0 0 -50 0 50 100 150 200 -200 0 200 400

-1 -1 1/[Guaiacol] (mM ) 1/[H2O2] (mM )

(a) (b)

Figure 9. Determination of ( kineticA) parameters of peroxidase from Turkish black radish ( R. sativus L.). ((B)A) Double reciprocal plot for guaiacol oxidation by the peroxidase: Assays were carried out at 4.5 mM H2O2 in 0.1 M sodium phosphate buffer (pH: 7.0) at 25°C. (B). Double reciprocal plot of activity vs. H2O2 concentration for the peroxidase: Assays were carried out at 45.5 mM guaiacol in 0.1 M sodium phosphate buffer (pH: 7.0) at 25°C.

(Table 2). As a conclusion, the present study showed that candidate for further studies such as in chemical peroxidase from Turkish black radish produce copious diagnostics. Also, it can be used for the applications such levels of POD, which was purified and characterized to as synthesis of various aromatic compounds, removal of homogeneity. The purified enzyme showed better thermal phenolics from waste waters and the removal of stability indicating its wider applications. The present peroxides from foodstuffs, beverages and industrial study also showed that this enzyme is an interesting wastes.

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