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Initial Submission

Orjinal Article

Partial Purification and Some Properties of Catalase from Dill (Anethum graveolens L.)
Gulnur ARABACI
2

Sakarya University, Faculty of Arts & Science, Department ofChemistry,54187, Sakarya,Turkey Received: Accepted: Published:

Abstract Catalase (CAT: EC 1.11.1.6) is a metalloenzyme which dismutes hydrogen peroxide to water and oxygen. It is widely distributed in animals, plants, all aerobic microorganisms. Most of work performed on this enzyme obtained from mammalian, bacteria and fungal sources as there is less work about plant catalases. In this work, partial purification of catalase from dill ( Anethum graveolens L.) and the kinetic properties were investigated. For the purification, enzyme was extracted from dill with phosphate buffer and centrifuged. Then (NH4)2SO4 precipitation was performed to the extracted enzyme and was dialyzed. After dialysis, the extract was loaded and eluted from a sephadex G200 column with phosphate buffer. The overall partial purification was about 5.5-fold. A temperature of 4C was maintained during the purification process. Enzyme activity was spectrophotometrically measured at 240 nm. The optimal pH and temperature were determined as 7.0 and 30C. In addition, KM and Vmax values were determined with H2O2 by means of Lineweaver-Burk plots. KM and Vmax of dill catalase was 24.0 mM and 3333.33 U/mL respectively. The effect of citric acid, KCN, NaN 3, NaCl and CuSO4 on dill catalase activity was investigated and the results showed that KCN and NaN3 had good inhibitory effect.
Key words: Purification, Catalase, Dill, Properties

Corresponding Author: G.Arabaci, e-mail: garabaci@sakarya.edu.tr, Phone:+902642956048, Fax:+ 902642955950

INTRODUCTION Catalase (CAT, H2O2:H2O2 oxidoreductase; EC 1.11.1.6) by scavenging hydrogen peroxide to water and oxygen is an important enzyme of cell defense mechanisms against oxidative stress in plants (Dat et al. 2003; Foyer and Noctor 2000). Catalase is widely distributed in a variety of organisms, including animals, plants, all aerobic microorganisms and some anaerobic organisms (Abassi 1998; Bailly et al. 2004). Different organisms have shown different catalas activities. In plants, multiple isoforms of the enzyme are usually present, and they are expressed in different tissues and developmental stages. In green leaves a majority of CAT activity is found in peroxisomes (Foyer and Noctor 2000; Scandalioset al. 1997). CAT scavenges H2O2 generated during -oxidation of the fatty acids, electron transport in mitocondria and photorespiratory oxidation (Havir and Mchale 1987; Kunce and Trelease 1986). Catalase is the most efficient enzyme as an antioxidative enzyme which lowers, hydrogen peroxide or superoxide to accumulate to toxic levels in plant growth (Bowler et al. 1992; Brenan and Frenkel, 1977). Catalases are predominantly heme-containing tetrameric proteins (Switala and Leuwen 2002). Catalase enzyme has been isolated, purified and characterized from many eucaryot and procaryot organisms. A comparison of CATs shows a wide range of activities and kinetic properties Catalase has been reported to exist in many plants, such as spinach, maize, cotton, sunower, tobacco, van apple, and parsley (Garcia et al. 2000; Mullen and Gifford 1993; Yoruk 2005; Lokman et

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al. 2007). Science various catalases purified so far had shown different characteristics and also catalases from greening pumpkin cotyledons had been purified and charactherized with low catalytic activity (Yamagughi et al. 2005). The ethanolic extraction of dill flower and seeds were investigated and showed high antioxidant activity by Shyu (Shyu et al. 2009). However, the isolation, purification and kinetic properties of catalase from dill (Anethum graveolens L.) have not been reported in literature yet. The aim of the present study was to characterize the CAT from dill (Anethum graveolens L.) and investigate the effects of various compounds to the enzyme activity. MATERIAL AND METHODS Materials and Chemicals
Dill (Anethum graveolens L.) used in this study was obtained from Sakarya region and stored at 20C until used. Hydrogen peroxide, polyviniylpolypyrolidone, Sephadex G200, (NH4)SO4 and other chemicals were obtained from Sigma Chemical Co., St. Louis, MO.

G. Arabaci
activity was estimated by decreased in absorbance of H2O2 at 240 nm. Effect of pH The effect of pH on CAT activity obtained at different pH values (pH 3.0-9.0). The optimum pH of dill CAT was determined. Effect of temperature: The effect of temperature on CAT activity obtained at different temperature values (20-80oC) and the optimum temperature of dill CAT was determined. Kinetic Determinations For determination of Michaelis constant (KM) and maximum velocity (Vmax) values of the enzyme, CAT activity was measured with H2O2 at varying concentrations (2-30 mM) at 240 nm. Kinetic constants as Vmax and KM values were determined by Lineweaver-Burk graphs (Fig. 4). Effect of inhibitors Effect of inhibitors potassium cyanide (KCN), citric acid, NaCl, NaN3 and CuSO4 were used as inhibitor. RESULTS AND DISCUSSION In aerobic organisms and plants, oxygen is an essential element, but it can be reduced and form reactive oxygen species (ROS) to be a danger and limiting factor in growth and development of plants (Scandalios et al. 1997). To minimize these ROS molecules, all aerobic organisms including plants, have evolved various enzymatic and nonenzymatic defense mechanisms. Catalase is one of several antioxidant defence enzymes (others include superoxide dismutase, peroxidases, glutathione) that catalyzes the dismutation of hydrogen peroxide to water and oxygen. Catalase (H2O2:H2O2 oxidoreductase; EC 1.11.1.6) is a tetrameric heme-containing enzyme found in all aerobic organisms, is known to play a key role in protecting cells against oxidative stress (Chaudiere and Ferrari-Iliou 1999). In this paper, catalase was partially purified from dill and the kinetic properties were studied. Typical results of the partial purifications procedure of CAT from dill, 15 g, are given in Table 1. After extraction, several precipitations with solid (NH4)2SO4 between 020%, 2040%, 4050%, 5060%, and 6080% were tested to find the appropriate saturation point. In conclusion, CAT activity of the precipitate of 3060% (NH4)2SO4 saturation was found to be 52.63% purification, and this saturation point was used all the extraction processes. After ammonium sulfate precipitation, the dialyzed enzyme extract was loaded onto Sephadex G200 gel filtration column and eluted with 50 mM phosphate buffer (pH 7.0). As

Extraction and purification 15 g of dill was obtained from local Sakarya region. After that samples were added to 10 ml 50mM sodium phosphate buffer (pH; 7.0), 0.3 g polyvinylpolypyrolidone (PVPP), and extraction was prepared. The mixture was homogenized with blender. After the filtrate was centrifuged at 14.000 g for 30 min and supernatant was collected (Gholamhoseinian 2006). Extraction was fractionated with (NH4)2SO4, solid (NH4)2SO4 was added to the supernatant to obtain 80% saturation. The mixture was centrifuged at 14,000 g for 30 minutes and the precipitate was dissolved in a small amount of phosphate buffer and then dialyzed at 4C in the same buffer for 24 h with three changes of the buffer during dialysis. The dialyzed enzyme extract was centrifuged and loaded onto Sephadex G200 column (1X10 cm) previously equilibrated with extraction buffer, and washed with the same buffer to remove unbound proteins. The eluate was used as the CAT enzyme source in the following experiments. CAT active fractions were pooled as purified CAT for characterization. Determination of protein The amount of CAT was performed according to method of Bradford with bovine serum albumin as standard (Bradford, 1976). Enzyme assay Catalase (CAT) activity was determined at 25oC according to Aebi (Aebi 1984). The reaction mixture contained 30 mM H2O2 in a 50 mM phosphate buffer pH 7.0, and 0.1 ml enzyme in a total volume of 3 ml. Catalase (CAT)

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a result, CAT enzyme from dill was purified 5.5-fold with a yield 18.18%. Table 1 Partial purification of catalase from dill (Anethum graveolens L.) Purification Step Crude Extract (NH4)2SO4 Sephadex G200 Protein (mg/ml) 82 90 38 Activity (Units/ml) 876 1828 2225 Total Activity (Units) 437880 274185 111245 Specific Activity (Units/mg) 10.68 20.31 58.55 Purification (fold) 1.0 1.9 5.5

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Yield (%) 100 52.63 18.18

Relative Activity (%)

The effects of temperature between 10 and 70C on dill CAT activity were assayed; results are shown in Figure 1. Maximum CAT activity was found to be at 30C. The optimum temperature we determined was similar to that given in previous studies (Ozturk et al. 2007). Some scientists found that the optimum temperature was 30 and 50C in chard and Van apple, respectively (Dincer et al. 2001; Yoruk et al. 2005).
100

100

80

60

40

80

20

Relative Activity (%)

60

0 0 2 4 pH 6 8 10

40

20

Figure 2 Effect of pH on the activity of dill catalase.

0 0 20 40 60 80 100

0,006

T emperature o C

The optimum pH for dill CAT activity was determined at different pH values as shown Figure 2. As indicated, the optimal pH for dill CAT was found to be 7.0. The active pH of cabbage CAT had shown to be around 6 to 8 with is similar to our results (Gholamhoseinian 2006). The optimal pH for most CAT has been shown around 6.8 to 7.5 (Aebi 1984). The affinity of dill CAT towards its own substrate was determined at optimum pH and temperature with different H2O2 concentration by the Lineweaver-Burk plots (Figure3). KM and Vmax values were calculated 24.0 mM, and 3333.33 U/mL for H2O2, respectively.

1/V(EU/mL)

Figure 1 Effect of temperature on the activity of dill catalase.

0,004

0,002

0 -0,1 0 0,1 0,2 0,3 0,4 0,5 0,6

1/[H2 O2 ] mM

Figure 3 Lineweaver-Bruk double reciprocal plot at different H2O2 concentration for dill catalase.

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The effect of time on the activity of dill catalase was also measured at room temperature. After 72 hours, only 10% of the activity was remained.
120 100
Relative Activity (%)

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Table 2 Effects of various compouns on CAT from dill (Anethum graveolens L.) Inhibitors Concentration (mM) 0.5 1 3 5 0.5 1 3 5 0.5 1 3 5 0.5 1 3 5 0.5 1 3 5 Relative activity (%) 93.4 74.2 52.0 17.2 97.5 89.8 82.5 78.1 52.4 28.2 12.5 2.5 101.0 102.3 104.1 106.2 100.1 99.8 990 98.3

KCN

80 60

Citric acid
40 20 0 0 20 40 60 T ime (Hours) 80 100 120

NaN3

CuSO4
Figure 4 Effects of time on the activity of dill catalase.

NaCl

Effects of various compounds on dill catalase activity were measured. The inhibitory effect of citric acid, potassium cyanide, NaCl, NaN3 and CuSO4 at different concentrations (0,5, 1, 3, 5mM) on CAT activity were estimated at 25oC and pH 7.0 by using fixed H2O2 as a substrate (Table 2). Citric acid, NaCl and CuSO4 had almost no effect to dill CAT activity up to 5 mM inhibitor concentration. However, potassium cyanide and NaN3 had very high inhibitory effect on the catalase activity. The results can be suggested that potassium cyanide and NaN3 at 0,5-5 mM concentration can be used as inhibitors to minimized the catalase activity when it is desired. REFERENCES
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Mullen RT, Gifford DJ, 1993. Purification and characterization of catalase from Loblolly Pine ( Pinus taeda L.) Megagametophytes. Plant Physiol. 103: 477483 Pery ME, 1984. Catalase an old enzyme with role? Can. J.B.ochem. Cell. Biol. 62: 1006-1014 Switala J, Leuwen PC, 2002.Diversity of properties among catalases. Arch.Biochem. Biophys. 401: 145-154 Scandalios JG, Guan L, Polidoros AN, 1997. Catalases in plants: gene structure, properties, regulation and expression, in: J.G. Scandalios (Ed.), Oxidative Stress and the Molecular Biology of Antioxidant Defences Cold Spring. Harbor Laboratory Pres. N.Y. 343-406. Shyu YS, Lin JT, Chang YT, Chiang CJ, Yang DJ, 2009. Evaluation of antioxidant ability of ethanolic extract from dill (Anethum graveolens L.) flower. Food Chemistry. 115: 515521 Yamagughi J, Nishimura M, Akazawa T, 2005. Purification and characterization of heme-containing low-activity form of catalase from greening pumpkin cotyledons. European Journal of Biochemistry 159: 315322 Yoruk IH, Demir H, Ekici K, Sarvan A, 2005. Purification and properties of catalase from Van Apple (Golden Delicious). Pak. J. Nutr. 4: 8-10

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