PURIFICATION AND CHARACTERIZATION OF PEROXIDASE FROM SWEET GOURD

Protein & Peptide Letters, 2008, 15, 000-000 1

0929-8665/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd.

Purification and Characterization of Peroxidase from Cauliflower (Bras-sica oleracea L. var. botrytis) Buds

Ekrem Köksal and lhami Gülçin*

Atatürk University, Science and Arts Faculty, Department of Chemistry, 25240-Erzurum, TR-Turkey

Abstract: Peroxidases (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) are part of a large group of enzymes. In this study, peroxidase, a primer antioxidant enzyme, was purified with 19.3 fold and 0.2% efficiency from cauliflower (Brassica oleracea L.) by ammonium sulphate precipitation, dialysis, CM-Sephadex ion-exchange chromatography and Sephadex G-25 purification steps. The substrate specificity of peroxidase was investigated using 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid) (ABTS), 2-methoxyphenol (guaiacol), 1,2-dihydroxybenzene (catechol), 1,2,3-trihyidroxybenzene (pyrogallol) and 4-methylcatechol. Also, optimum pH, optimum temperature, optimum ionic strength, stable pH, stable temperature, thermal inactivation conditions were determined for guaiacol/H2O2, pyrogallol/H2O2, ABTS/H2O2, catechol/H2O2 and 4-methyl catechol/H2O2 substrate patterns. The molecular weight (Mw) of this enzyme was found to be 44 kDa by gel filtration chromatography method. Native polyacrylamide gel electrophoresis (PAGE) was performed for isoenzyme determination and a single band was observed. Km and Vmax values were calculated from Lineweaver-Burk graph for each substrate patterns.

Keywords: Cauliflower, Brassica oleracea, peroxidase, enzyme purification, chromatography.

INTRODUCTION

Peroxidase (POD) is a heme protein, which is a member of oxidoreductases [E.C.1.11.1.7] and catalyses the oxidation of a wide variety of organic and inorganic substrates in the presence of hydrogen peroxide [1,2]. Peroxidases are widely distributed in living organisms including microorganisms, plants and animals. POD and catalase are two major systems for the enzymatic removal of H2O2 and peroxidative damage of cell walls is controlled by the potency of antioxidative peroxidase enzyme system [3,4]. This enzyme is one of the key enzymes controlling plant growth and development. It takes place in various cellular process including construc-tion, rigidification and eventual lignifications of cell walls, protection of tissue from damage and infection by patho-genic microorganisms [5,6]. This enzyme participates in the formation of lignins in the secondary cell walls during nor-mal growth [7,8] and in the formation of phenolic polymers such as lignins, suberins, etc. when plants are infected or wounded [9]. POD is also widely used as an important rea-gent for clinical diagnosis and microanalytical immunoassay. Some applications for POD have been suggested in the me-dicinal, chemical and food industries [10]. It has been re-ported that peroxidase has been used for biotransformation of organic molecules [11-13]. Because of its broader cata-lytic activity, a wide range of chemicals can be modified using POD. Also, it can be used for the applications such as synthesis of various aromatic compounds, removal of phe-nolics from waste waters and the removal of peroxides from foodstuffs, beverages and industrial wastes [14]. POD is also

Address correspondence to this author at the Atatürk University, Science and Arts Faculty, Department of Chemistry, TR-25240, Erzurum- Turkey; Tel: +90 442 2314444; Fax: +90 442 2360948; E-mails: [email protected]; [email protected]

related to quality of plant commodities, particularly the fla-vour, in both raw and processed foods. POD activity is also correlated to fruit ripening as has been shown in a number of cases and it is also involved in enzymatic browning, either or together with polyphenol oxidase activity. A more precise understanding of the implication of POD in these mecha-nisms is an essential step towards a more efficient control of these undesirable reactions, particularly in heat-processed products, which frequently contain residual peroxidase activ-ity [15,16].

In Turkey, cauliflower is cultivated mainly in the East Black Sea coast. In this study we report the purification and kinetic characterization of POD from cauliflower.

MATERIALS AND METHODS

Plant Materials

Fresh cauliflower (Brassica oleracea L.) buds were ob-tained from a local market in Erzurum, Turkey. It was washed, drained, packed in polyethylene bags and stored at -83oC until using.

Preparation of Cauliflower Homogenate

The homogenate preparation procedures for POD were adapted from the methods described by Sakharov and co-workers [17]. For this purpose, 20 g cauliflower were taken from frozen storage (-83oC) and ground in a mortar in the presence of liquid nitrogen. This powder then mixed with 50 mL of phosphate buffer (pH 7.0, 0.1 M) and subsequently collard slurry was centrifuged at 15.000xg for 60 min at 4oC [18]. The buffer composition was as follows: 0.1 M phos-phate buffer; PVP (0.05%); pH 7.0. The pellet was dis-carded.

wasim

Final

2 Protein & Peptide Letters, 2008, Vol. 15, No. 4 Köksal and Gülçin

Ammonium Sulphate Precipitation and Dialysis

The crude extract was subjected to ammonium sulphate fractionation and the precipitate in the 20-70% saturation range was collected by centrifugation 60 min at 15.000xg. The precipitate was suspended in about 2 mL phosphate buffers (pH 7.0, 0.1 M) and dialyzed 12 h at 4oC against 1 L above buffer for further use.

Preparation of CM-Sephadex A-50 Ion Exchange Chro-

matography Material

Briefly, 3.5 gram dried CM-Sephadex A-50 (Sigma) was dissolved in 100 mL distilled water and incubated in a 90oC water bath for 5 hours. Following cooling to the room tem-perature, this slurry was mixed with 100 mL NaOH (0.5 M) and was allowed to stand for 1 hour. Afterwards, the super-natant was decanted and the exchanger was washed with distilled water until the effluent is at neutral pH. Then, the exchanger was stirred in 100 mL 0.5M HCl and allowed to stand for an additional 1 hour. Subsequently, the exchanger was washed with distilled water until the effluent was at pH 7.0. Finally, the exchanger was suspended in 0.1 M phos-phate buffer (pH 6.5) then packed in a column (3x30 cm) and washed and equilibrated with the same buffer. The flow rates for washing and equilibration were adjusted by peristal-tic pump as 40 mL/h [19].

Purification of Peroxidase by CM-Sephadex A-50 Ion

Exchange Chromatography

Dialyzed cauliflower sample was loaded onto CM-Sephadex A-50 column previously equilibrated column and the gel was washed with 1 L of phosphate buffer (pH 7.0, 0.1 M). Bound proteins were eluted with a gradient of (250 mL) 0-1 M NaCl in 10 mM phosphate buffer pH 7.0 at flow rate of 15 mL/h by using gradient mixer apparatus (Pharmacia Fine Chemicals). Eluates were collected as 3 mL fractions and each of their activity and absorbance were separately measured at 470 nm and 280 nm respectively [20]. Active fractions were pooled and kept at +4oC until use.

Purification and Concentration of Peroxidase by Se-

phadex G-25

Briefly, 20 mL of active fractions were obtained from CM-Sephadex A-50 column chromatography were mixed with 4 g of Sephadex G-25 in a centrifuge tube. Then the mixture was centrifuged for 3 min at 1000xg (MSE Mistral 2000, U.K). The upper layer of solution (2.5 mL) was taken and the POD enzyme activity measured at 470 nm in a spec-trophotometer by using guaiacol substrate. For determining specific activity, POD activity and quantitative protein measurements were carried out. Protein contents were de-termined by the protein dye-binding Bradford’s method [21].

Determination of Molecular Weight of Peroxidase by Sephadex G-100 Gel Filtration Chromatography

For molecular weight of POD, a purified and concen-trated enzyme aliquot was fractionated by gel filtration chromatography. In gel filtration chromatography, a column with 100 mL bed volume was prepared using Sephadex G-

100 and equilibrated with phosphate buffer (pH 7.0, 0.1 M). The purified and concentrated enzyme solution was passed through the column. The elution rate was adjusted to 4 mL/h. The eluates were collected in tubes as 3 mL volumes. The elution process was continued until the absorbance values reached to zero point at 280 nm. Qualitative protein determi-nation was done at 280 nm on the eluates obtained and POD activity was measured in the eluates showing absorbance at 470 nm.

Native PAGE Electrophoresis

Native polyacrylamide slab gel electrophoresis (PAGE) was performed according to Laemmli’s procedures [22] un-der native conditions (i.e. without sodium dodecyl sulfate) for separating POD isoenzymes under natural condition [23]. The experiment was conducted in a cold room at +4oC with the electrode buffer Tris/glycine (pH 8.3) using 3% stacking gel and 10% separating gels. The enzyme samples were loaded on to each space of the stacking gel at different amount. Initially, an electric current on 80 V was applied until the bromophenol dye extended into the separating gel and then increased to 150V for 5-6 h until the tracking dye migrated to 1 cm from the bottom. After running, gels were incubated in 45 mM guaiacol and 22.5 mM H2O2 in 100 mM phosphate buffer (pH:7) at 37oC until appearance of the en-zyme bands and then photographed (Fig. 3).

Peroxidase Activity Assay

The POD activity in the cauliflower sample was meas-ured using various substrates such as ABTS, catechol, 4-methyl catechol, pyrogallol and guaiacol. When studying substrate specificity of POD, the activity was measured un-der optimal conditions determined for each substrate. Tem-perature was controlled using a circulating water bath with a heater/cooler (Grant LTD 6G -20 to 100oC, England). Initial rates of free radical formation for substrates were monitored at maximum wavelength of each substrate. The changes in absorbance were read for 3 min using a double beam UV-VIS spectrophotometer (CHEBIOS s.r.l.). The following wavelengths were used in the assays: at 415 nm for ABTS and pyrogallol, 290 nm for catechol and 4-methyl catechol and 470 nm for guaiacol, which are substrates of POD. As-say methods for various substrates were described previously [24]. Briefly, an aliquot of enzyme sample (25 L) was added to a mixture of 1 mL 22.5 mM H2O2, 1mL 45 mM guaiacol, and final volume of this mixture was adjusted to 3 mL by addition of phosphate buffer (pH:7.0, 0.1 M). The change in the absorbance at above wavelength monitored for 3 min at 20oC. One unit of peroxidase activity was defined as 0.01 A470 per min [20,25].

Qualitative and Quantitative Protein Determination

Qualitative protein determination was done at 280 nm on the eluates obtained and POD activity was measured in the eluates showing absorbance at 280 nm. Quantitative protein determination was achieved by absorbance measurements at 595 nm according to Bradford’s method [21], with bovine serum albumin as standard.

Purification and Characterization of Peroxidase from Cauliflower Protein & Peptide Letters, 2008, Vol. 15, No. 4 3

Kinetic Studies

Optimum pH Profile

The optimum pH value for the activity of the POD was found by assaying enzyme activity at different pH levels. The assay was carried out by taking buffers of different pH such as (pH 3.0-4.5, 0.1 M sodium acetate; pH 4.5-7.5, 0.1 M sodium phosphate; pH 8.0-9.0, 0.1 M Tris-HCl) pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 sepa-rately in an assay mixture. All assays were made with hydro-gen peroxide and various reducing substrate concentrations [25].

pH Stability Profile

When checked for stability pH using guaiacol, pyrogal-lol, ABTS, catechol and 4-methyl catechol as H-donor. POD from cauliflower showed maximum activity at pH ranging from 4-7.5. Similarly, the commercial horseradish peroxi-dase showed highest activity at pH ranging from 4 to 5 and POD from red beet hairy root showed maximum activity at pH ranging from 5 to 6. The enzyme was stable over a wide range of pH from 4 to 9 exhibiting highest stability at pH 4 and 8.5 (Table 2).

The Effect of Temperature

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 the mixture as rapidly as possible. For determining optimum temperature ranges of the enzyme for each of the above mentioned five substrates, activity was measured at different temperatures in the range from 0-90oC. In order to determine the thermal stability of the POD, we used above mentioned substrates. The purified enzyme was studied at 30, 40, 50, 60, 70, 80, and 90oC. For the study, 1 mL of 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 the required time interval, the enzyme was cooled in an ice bath and brought to room tem-perature. Under optimal conditions, 0.1 mL heated enzyme extract was mixed with substrates and buffer, and residual POD activity was determined spectrophotometrically. The percentage residual POD activity was calculated by compari-son with unheated enzyme [26,27].

The POD extracted at acidic and neutral pH showed neg-ligible inactivation up to 50oC with almost 95% of the activ-ity being retained even after 40 min. However, the POD of basic pH was very sensitive to temperature with 50% loss at 50oC in 40 min, with the total loss of activity at 60oC [28]. At the latter temperature (60oC) the acidic and neutral PODs retained more than 70% of the activity up to 40 min with complete inactivation at 70oC. As can see in Fig. 4, our re-sults showed that POD purified from cauliflower was more stable at temperature ranged in 30-50oC. Also, the extent of the effect depends on the chemical nature of the studied sub-strate. In the case of ABTS, the POD activity declined after ten minute. These results suggest that the studied substrates having different chemical structures react with different re-gions of the active site of the POD.

Effect of Ionic Strength

The effect of ionic strength on the enzyme was studied for each substrate using different concentrations of buffers (0.1-1 M). Table 2 showed that the POD activity varied slightly with the buffer concentration range from 0-1 M. In general, POD activity was maximal at 1 M of buffer concen-tration.

Substrate Specificity

Under optimal conditions, the efficiency of catalytic oxi-dation of the substrates such as guaiacol, pyrogallol, ABTS, catechol, and 4-methyl catechol by hydrogen peroxide in the presence of POD was evaluated. Km and Vmax values were calculated for peroxidase reactions with each of the five sub-strates, using the Lineweaver-Burk transformation of the Michaelis-Menten equation. Table 2 shows the kinetic con-stants of POD purified from cauliflower. Km and Vmax values were determined for each of guaiacol/H2O2, ABTS/H2O2, pyrogallol/H2O2, catechol/H2O2 and 4-methyl catechol/H2O2 substrate pairs. For this, the enzyme activity was measured at five different concentrations of pyrogallol or guaiacol while H2O2 concentration was constant. In addition, this measure-ment was performed at a constant concentration of pyrogal-lol, guaiacol, ABTS, catechol or 4-methyl catechol whilst five different H2O2 concentrations were used. Km and Vmax values were calculated from the plot of 1/V versus 1/[S] by the method of Lineweaver and Burk [29].

RESULTS AND DISCUSSION

CM-Sephadex A 50 Ion Exchange Chromatography

The dialyzed enzyme extract was applied to a CM-Sephadex A 50 ion exchange chromatography and bound proteins were eluted with a linear gradient of 0-1 M NaCl in 100 mM phosphate buffer (pH:7.0) at flow rate of 15 mL/h. Eluates were collected as 3 mL fractions and each of their activity and absorbance were separately measured at 420 nm and 280 nm respectively (Fig. 1). Active fractions were pooled and kept at +4oC until use.

Figure 1. Anion exchange chromatography of POD from cauli-flower (Brassica oleracea L.) buds: Elution profile of unbound fraction from CM-Sephadex A-50 obtained in 0.1 M sodium phos-phate 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 on the right hand y-axis. NaCl gradient started with beginning of elution.


Purification and Concentration of Peroxidase by Se-

phadex G-25

Briefly, 20 mL of active fractions were obtained from CM-Sephadex A-50 column chromatography were mixed with 4 g of Sephadex G-25. The mixture was centrifuged for 3 min at 1000xg. Then the upper layer of solution (2.5 mL) was taken and the POD enzyme activity measured at 470 nm in a spectrophotometer by using guaiacol substrate. By ap-plying this method, 19.33-fold purification was obtained (Table 1).

Molecular Weight Determination

The molecular weight of the POD was determined by gel filtration chromatography according to Andrews’ method [30]. The void volume of the column was determined with Blue Dextrane 2000. -galactosidase (116.000), bovine se-rum albumin (66.000), chicken serum albumin (45.000) and carbonic anhydrase (25.000) were used as standard proteins (Sigma: MW-GF-200). A Log MW-Elution volume graph obtained. Molecular weight of POD was calculated by using the following formula obtained standard graph.

0.9991)(R 2.1564 olume][Elution v x 0.0065- M Log 2

W =+=

Then, elution number of purified peroxidase was inserted into equation obtained from above graph. Thus, Mw of puri-fied peroxidase was calculated as 44 kDa. Gel filtration chromatography profile of peroxidase from cauliflower buds was shown in Fig. 2. Similarly, the molecular mass of the purified cationic peroxidase from Korean radish seeds (Rap-hanus sativus) was estimated to be about 44 kDa on SDS-PAGE [31]. However, Scialabba and co-workers showed four peroxidase isozymes with molecular weight of 98, 52.5, 32.8 and 29.5 kDa were expressed in maturing radish seed [32]. On the other hand, Gülçin and Yıldırım purified and characterized peroxidase from Brassica oleracea with 95 kDa molecular weight [13].

Native PAGE Electrophoresis

Native polyacrylamide slab gel electrophoresis (PAGE) was performed under native conditions such as without so

Figure 2.Gel filtration chromatography profile of peroxidase from cauliflower (Brassica oleracea L.) buds.

dium dodecyl sulphate and low temperature (+4oC) for sepa-rating POD isoenzymes under natural condition. After elec-trophoresis, the gel was incubated in peroxidase staining solution containing pyrogallol/H2O2 substrate pairs. As can be seen in Fig. 3, one isoenzyme was found by using the substrate pairs.

Optimum pH

The optimum pH value for the activity of the POD was found by assaying enzyme activity at different pH levels. As can be seen in Table 2, the optimal pH of POD for guaiacol substrate was determined to be 5.0 using 0.1 M sodium phosphate buffers. This value for ABTS and catechol sub-strates was found to be 4.0 using 0.1 M sodium acetate buffer and 7.0 using 0.1 M sodium phosphate buffer, respec-tively. For pyrogallol and 4-methyl catechol substrates was found as 7.5 using 0.1 M sodium phosphate buffer.

Stable pH

To be able determine the stabile pH of peroxidase en-zyme; activity of it was followed in three different buffers, have a pH range of pH 4.5 to pH 9 at 0.5 pH intervals, for 10 days. As it can be seen in Table 2; POD was more stable at

Table 1. Levels of Purification of Cauliflower (Brassica oleracea L.) Buds Peroxidase Obtained After the Application of Different

Purification Steps Leading to the Improvement in the Activity of Enzyme

Purification steps Total

volume (mL)

Enzyme

activity

(EU/mL.min)

Total enzyme

activity

(EU/mL.min)

Protein

(mg/mL)

Total Protein

(mg)

Specific

activity

(EU/mg)

Yield (%) Purification

fold

Homogenate 100 21960 2196000 0.909 90.9 24158.4 100 1

(NH4)2SO4 precipita-

tion

12 69560 834720 2.121 25.452 32795.9 28 1.358

Dialysis 12.4 59440 737056 1.393 17.2 42670.5 19 1.766

CM-Sephadex ion

exchange chromatog-

raphy

20 22800 456000 0.097 1.94 235051.6 2 9.7

Sephadex-G-25 2.5 38760 96900 0.083 0.2 466987.9 0.2 19.33


pH ranged 8-9 in 0.1 M Tris/HCL buffer at the end of this incubation period.

Figure 3.Native polyacrylamide gel electrophoresis zymogram of peroxidase from cauliflower (Brassica oleracea L.) buds (Line 1: 30 g POD, Line 2: 20 g POD, Line 3: 10 g POD, Line 4: 20 g POD).

Optimum Temperature

For determining the optimum temperature values of the enzyme, POD activity was measured at different tempera-tures in the range from 5 to 90oC for 5 minutes under optimal pH and buffer concentration for each substrate. Once tem-perature equilibrium was reached, enzyme was added and the reaction was followed spectrophotometrically at constant temperature at given time intervals. As seen in Fig. 4, the optimum temperature of peroxidase enzyme was determined to be 25oC for pyrogallol, 30oC for guaiacol and ABTS sub-strates, 45oC for 4-methyl catechol, 50oC for catechol. The enzyme activity was too low at 80oC, hence above this tem-perature was not studied.

Figure 4.The effect of temperature on the peroxidase activity from cauliflower (Brassica oleracea L.) buds.

Thermostability of the Purified POD

Temperature effects on the POD were carried out by measuring the residual activity after incubating 1 mL of the enzyme at different temperatures in a water bath in the range from 10 to 60oC under optimal pH and buffer concentration for each substrate (Fig. 5). Under optimal conditions, 0.25 mL heated enzyme extract was mixed with substrates and buffer, and residual POD activity was determined spectro-photometrically. The percentage residual POD activity was calculated by comparison with unheated enzyme.

Kinetic Studies

To be able compare substrate specificity, KM and Vmax

values were determined for each of guaiacol/H2O2, ABTS/H2O2, pyrogallol/H2O2, catechol/H2O2 and 4-methyl catechol/H2O2 substrate pairs. For this, the enzyme activities were measured at five different concentrations of substrates while H2O2 concentration was constant. KM and Vmax values were calculated from Lineweaver-Burk graphs of the results obtained from above experiments. Enzyme had Km values of 141.64, 1.1, 2.01, 3.41 and 8.19 mM for above mentioned substrates, respectively. On the other hand, enzyme had Vmax values of 7500, 4400, 590, 300, and 1340 EU/mL.min for each substrate respectively (Table 2). These results showed

Table 2. Optimum pH, Optimum Temperature, Optimum Ionic Strength, Stable pH, Thermal Stabilization and Substrate Speci-

ficity of Peroxidase from Cauliflower (Brassica oleracea L.) Buds

Substrate Guaiacol Pyrogallol ABTS Catechol 4-Methyl catechol

Optimum pH 5 4 7.5 7 7.5

Optimum temperature (oC) 30 30 25 50 45

Optimum ionic strength (M) 1.0 0.3 1.0 0.9 0.7

Thermal stabilization (oC) 40 40 20 40 40

Stable pH 8.5 8.5 9.0 8.5 8.0

Km (mM) 141.64 1.1 2.01 3.41 8.19

Vmax(EU/mL.min) 7500 4400 590 300 1340


that enzyme had the most affinity to pyrogallol substrate. These results suggest that among ABTS, pyrogallol and guaiacol, the most suitable compound, as H-donor is pyro-gallol for the peroxidase enzyme from cauliflower buds.

CONCLUSION

The present study has shown that peroxidase from cauli-flower produce copious levels of POD, which was purified and characterized to homogeneity. The purified enzyme showed better thermal stability indicating its wider applica-tions. The present study has also shows that this enzyme an interesting candidate for further studies such as in chemical diagnostics. Also, it can be used for the applications such as synthesis of various aromatic compounds, removal of phe-nolics from waste waters and the removal of peroxides from foodstuffs, beverages and industrial wastes. Cauliflower can use as a potential POD source.

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Received: October 24, 2007 Revised: November 21, 2007 Accepted: January 14, 2008

PURIFICATION AND CHARACTERIZATION OF PEROXIDASE FROM SWEET GOURD

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