Purification and Characterization of Peroxidase from Sweet Gourd (Cucurbita moschata Lam. Poiret)

Abstract

Peroxidase (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) is an oxidoreductase enzyme found in many fruits and vegetables. This enzyme was purified from sweet gourd (Cucurbita moschata Lam. Poiret) by ammonium sulphate precipitation and CM-Sephadex ion-exchange chromatography. Furthermore, optimum pH, optimum temperature, optimum ionic strength, stable pH, and stable temperature conditions were determined as 7.2, 50°C, 0.4 M, 8.0, and 40°C, respectively. The molecular weight (M_W) of the enzyme was estimated to be 85 kDa by SDS-PAGE method. The values of K_m and V_max were calculated from the Lineweaver-Burk graph for guaiacol/H₂O₂ substrate patterns.

Keywords:

• Sweet gourd
• Peroxidase
• Purification
• Chromatography
• Electrophoresis

INTRODUCTION

Peroxidase (E.C.1.11.1.7) (POD) is widely distributed in higher plants and catalyses the oxidation of a wide variety of organic and inorganic substrates in the presence of hydrogen peroxide.[1, 2] POD is a major system for the enzymatic removal of H₂O₂, and peroxidative damage of cell walls is controlled by the potency of an antioxidative peroxidase enzyme system.[3] POD is found in many plant-based foods. The enzyme is highly specific to its peroxide substrate, of which H₂O₂ is the most common, but it has low specificity toward its hydrogen donor substrate.[4] In the presence of peroxide, PODs from plant tissues are able to oxidize a wide range of phenolic compounds, such as guaiacol, pyrogallol, chlorogenic acid, catechin, and catechol.[5] This enzyme is involved in many functions, such as control of elongation, defense mechanisms,[6] and lignifications.[4] POD participates in the formation of lignins in the secondary cell walls during normal growth[7] and in the formation of phenolic polymers, such as lignins, suberins, etc., when plants are infected or wounded.[8] On the other hand, POD plays roles in food quality, including deterioration of color and flavor.[9,10] In addition, it is involved in plant hormone regulation,[11] defence indoleacetic acid degradation during maturation, and senescence of fruits and vegetables.[12] POD is also widely used as an important reagent for clinical diagnosis and microanalytical immunoassay. Some applications for POD have been suggested in the medicinal, chemical, and food industries.[13] The enzyme is reported to exist in both soluble and membrane-bound forms. It can be found in vacuoles, tonoplast, plasmalemma, and inside and outside the cell wall, and it has a variety of functions.[14] POD have been purified and characterized from several fruits and vegetables, for instance, cauliflower buds,[15] turnip,[16] tea leaves,[17] and rice.[18] PODs of these different plants showed different characteristics, such as molecular weight, optimum pH, and substrate specificity. In this study, the aim was to characterize and purify POD from sweet gourd as a new source. The main objective of this study was to determine selected kinetic properties, such as optimum pH, optimum temperature, optimum ionic strength, stable pH, and stable temperature conditions of POD from sweet gourd.

MATERIALS AND METHODS

Plant Materials and Chemicals

Fresh sweet gourd (Cucurbita moschata Lam. Poiret) was obtained from a local market in Erzurum, Turkey. Then, it was washed, peeled, broken up, packed in polyethylene bags, and stored at −83°C until the enzyme extraction. All other chemicals of analytical grade were obtained from either Sigma (Sternheim, Germany) or Merck (Darmstadt, Germany).

Preparation of Sweet Gourd Extract

The procedures of extraction for POD were described by Köksal and Gülçin.[15] For the extraction, 20 g of sweet gourd was taken out of the frozen storage (-83°C) and ground in a mortar in the presence of liquid nitrogen. This powder was then mixed with 50 mL of phosphate buffer (pH 7.0, 0.1 M) and, subsequently, was centrifuged at 15,000× g for 60 min at 4°C.[19] The buffer composition was as follows: 0.1 M phosphate buffer; PVP (0.05%); pH 7.0. The pellet was discarded. Several precipitations with solid (NH₄)₂SO₄, between 0–10, 10–20, 20–30, 30–40, 40–50, 50–60, 60–70, 70–80, and 80–90%, were tested to find the proper saturation point. The precipitated POD was separated by centrifugation at 15,000× g for 60 min. The enzyme activity of the precipitate of 60–90% (NH₄)₂SO₄ saturation was found to be the highest, and this saturation point was used for all the extraction processes. The precipitate was suspended in about 2 mL of phosphate buffer (pH 7.0, 0.1 M) and dialysed for 12 h at 4°C against 10 mM of phosphate buffer (pH 7.0). This sample was used in the following parts of the study.

Preparation of CM-Sephadex A-50 Ion Exchange Chromatography Material

Briefly, 3.5 g of dried CM-Sephadex A-50 (Sigma) was dissolved in 100 mL of distilled water and incubated in a 90°C water bath for 5 h. Following cooling at room temperature, this slurry was mixed with NaOH (100 mL, 0.5 N). Then, it was allowed to stand for 1 h. Afterwards, the supernatant was decanted and the exchanger was washed with distilled water until the effluent had a neutral pH. Then, the exchanger was stirred in 100 mL HCl (0.5). Subsequently, the exchanger was washed with distilled water until the effluent reached pH 7.0. Finally, the exchanger was suspended in 0.1 M phosphate buffer (pH 8.5) and was then packed in a column (3 × 30 cm), washed, and equilibrated with the same buffer. The flow rates for washing and equilibration were adjusted by a peristaltic pump as 15 mL/h.[20]

Purification of Peroxidase by Cation Exchange Chromatography

The column was packed with CM-Sephadex A-50. It was equilibrated with 1 L of Tris-HCl buffer (pH 8.5, 0.1 M). Then, the dialysed enzyme extract was loaded onto the column and washed with equilibrating buffer. Retained proteins were eluted with a gradient of (250 mL) 1 M NaCl in 10 mM phosphate buffer at a flow rate of 15 mL/h by using a gradient mixer apparatus (Pharmacia Fine Chemicals, Uppsala, Sweden). Fractions of 5 mL were collected and activity and absorbance of each were separately measured at 470 and 280 nm, respectively. Active fractions were kept at +4°C for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method. The enzyme was purified with 12% yield, 10.2-fold after the CM-Sephadex A-50 purification step (Table 1).

Table 1 Levels of purification of sweet gourd (Cucurbita moschata L.) peroxidase obtained after the application of different purification steps leading to the improvement in enzyme activity

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	23	6660	153,180	0.804	18.5	8280	100	1
(NH4)2SO4 precipitation	4	7000	28,000	0.749	3	9333.3	18	1.127
CM-Sephadex ion exchange chromatography	15	1240	18,600	0.22	3.3	84,545.4	12	10.2

Determination of the Purity and Molecular Weight of Peroxidase by SDS-PAGE

The molecular weight of the POD was determined by SDS-PAGE using the method of Laemmli.[21] The following molecular weight markers used for electrophoresis were obtained from New England BioLabs, Frankflurt, Germany. MBP-β-galaktosidas (175 kDa), MBP-truncated-β-galactosidase (83 kDa), MBP-CBD (62 kDa), aldolase (47.5 kDa), and triosephosphate isomerise (32.5 kDa). Polyacrylamide gels containing SDS (1%) were prepared as stacking gel 3% and separating gel (8%).[14] The gel was photographed following the SDS-PAGE process and molecular weight was calculated. To do this, the graph of log M_W -R_f values of protein marker was prepared according to the results of SDS-PAGE.[22–24] Then, R_f value of purified enzyme was obtained from this graph.

Enzyme Activity Assay

The POD activity in the sweet gourd sample was measured using guaiacol as the substrate. Temperature was controlled using a circulating water bath with a heater/cooler (Grant LTD 6G −20 to 100°C, Cambridge, England). The changes in absorbance were read for 3 min using a double beam UV-VIS spectrophotometer (CHEBIOS s.r.l., Rome, Italy), and 470 nm was used as the wavelength for guaiacol substrate. The final mixture contained 25 μL of the enzyme sample, 1 mL of 22.5 mM H₂O₂, 1 mL of 45 mM guaiacol, and the final volume of this mixture was adjusted to 3 mL by addition of phosphate buffer (pH 7.0, 0.1 M). Guaiacol is commonly used for plant POD. Also, the other substrate, such as ABTS, is frequently used. In addition, this chromogenic substrate was used for ABTS radical scavenging activity as an antioxidant measurement method.[25,26] The change in the absorbance at 470-nm wavelength was monitored for 3 min at 25°C. One unit of peroxidase activity was defined as 0.01 ΔA₄₇₀ per min.

Qualitative and Quantitative Protein Determination

Quantitative protein determination was carried out by using the method of Bradford[27] with bovine serum albumin (BSA) as a standard. During the purification process, qualitative protein determination was measured with absorbance at 280 nm.[14]

Characterization

The POD activity was investigated in the range of pH 3–9 using the following buffers: 0.01 M acetate buffer, pH 3–4.5; 0.01 M phosphate buffer, pH 5–7.5; 0.01 M Tris/HCl buffer, pH 8–9. All indication was made with hydrogen peroxide and guaiacol.[15] To determine the stable pH value of the enzyme, POD activity was observed for 10 days. The process was carried out using three different buffers with a pH range between 3–9 at 0.5 pH intervals. The activity measurements were performed at room temperature.

The enzyme activity was measured at different temperatures. At a certain temperature, enzyme activity was determined by the addition of enzyme to the mixture as rapidly as possible.[28] The process was carried out by a circulatory water bath in a temperature range between 10 and 80°C. To determine the thermal stability of the POD, the process was studied at 30, 40, 50, 60, and 70°C. Thus, 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 to room temperature. Under optimal conditions, 0.1 mL of heated enzyme extract was mixed with substrates and buffer, and residual POD activity was determined spectrophotometrically. The percentage of residual POD activity was calculated by comparison with unheated enzyme.[29] The effect of ionic strength on the enzyme activity was studied using different concentrations of buffers (0.025–0.7 M).[26] Studies were carried out at a predetermined optimal pH.

Kinetic Studies

To determine K_m and V _max values of guaiacol and H₂O₂ substrates, the enzyme activities were measured using five different concentrations of the substrates. K_m and V _max values were calculated from the Lineweaver-Burk graphs, whose results were obtained by the above experiments. K_m and V _max values were calculated for peroxidase reactions with each of the two substrates using the Lineweaver-Burk transformation of the Michaelis-Menten equation.[30]

RESULTS AND DISCUSSION

CM-Sephadex A 50 Ion Exchange Chromatography

The dialyzed enzyme extract was subjected to CM-Sephadex A 50 ion exchange chromatography and bound proteins were eluted with a linear gradient of 0.1–1 M NaCl in 100 mM of phosphate buffer (pH 8.0) at a flow rate of 10 mL/h. Eluates were collected as 5-mL fractions and the activity and absorbance of each were separately measured at 470 and 280 nm, respectively (Fig. 1). As a result of this process, POD was purified in 10.2-fold with 12% yield (Table 1).

Figure 1 Cation exchange chromatography of POD from sweet gourd (Cucurbita moschata L.): elution profile of unbound fraction from CM-Sephadex A-50 obtained in 0.1 M sodium phosphate buffer (pH 8.0) as 5-mL fractions.

Molecular Weight Determination and Purity Control

The SDS-PAGE photograph of peroxidase from sweet gourd is shown in Fig. 2. Finally, the molecular weight of POD from sweet gourd (Cucurbita moschata Lam. Poiret) was calculated to be 85 kDa. Similarly, M_W for one of the POD isoenzymes from radish seed was calculated to be 98 kDa.[31] Moreover, M_W of peroxidase from Brassica oleracea was estimated to be 95 kDa by SDS-PAGE.[16] A single band was observed in SDS-PAGE zymograme of POD purified from sweet gourd (Cucurbita moschata Lam. Poiret).

Figure 2 Photograph of SDS-PAGE. Line 1: molecular weight markers. Line 2: the peroxidase from CM-Sephadex cation exchange chromatography. (Color figure available online.)

Figure 3 The effect of incubation period on the peroxidase activity from sweet gourd (Cucurbita moschata L.) at different pH values: (a) acetate buffers; (b) phosphate buffers; (c) tris/HCl buffers.

Characterization Studies

In order to determine the optimum temperature values of the enzyme, POD activity was measured at different temperatures in a range between 10 and 80°C. As seen in Table 2, the optimum temperature of peroxidase enzyme was determined to be 50°C. It was observed that the enzyme activity increased to 50°C, and after this point it started to decrease. The optimum pH value for enzyme activity was determined by measuring the enzyme activity at different pH values. As can be seen in Table 2, the optimal pH of peroxidase from sweet gourd was determined to be 7.2 using 0.1 M of sodium phosphate buffers.

Table 2 Optimum pH, optimum temperature, optimum ionic strength, stable pH, thermal stabilization, and substrate specificity of peroxidase from sweet gourd (Cucurbita moschata L.)

Kinetic and optimum properties
Optimum pH	7.2
Optimum temperature (°C)	50
Optimum ionic strength (M)	0.4
Stable pH	8.0
Stable temperature (°C)	40
For guaiacol Km (mM)	17.1
For guaiacol V max (EU/mL.min)	15,500
For H2O2 Km (mM)	3.8
For H2O2 V max (EU/mL.min)	15,889

To assign the stable pH value of the enzyme, enzyme activity was monitored in three different buffers for 10 days, and it was demonstrated that POD was more stable in a pH range between 8–9.in 0.1 M of Tris/HCL buffer at the end of this incubation period. The results are shown in Figs. 3a–c. The results are in parallel with those obtained from POD from cauliflower.[15] Temperature effects on the POD were determined by measuring the residual activity after incubating 1 mL of the enzyme at different temperatures in a water bath at 30, 40, 50, 60, and 70°C and the relative results are given in Fig. 4. The results of the ionic strength study revealed that the salt concentration affects POD activity. POD activity was measured at 0.4 M of buffer concentration as maximum value. After this concentration, there was a linear decrease in enzyme activity. The results of this study are given in Table 2.

Figure 5 Thermal inactivation change of the peroxidase activity from sweet gourd (Cucurbita moschata L.).

Figure 5 Kinetic behavior of the two substrate reactions for sweet gourd (Cucurbita moschata L.) POD: (a) plot of guaiacol substrate; (b) plot of H₂O₂ substrate.

Kinetic Studies

To be able to determine the substrate specificity of the enzyme to guaiacol and H₂O₂ substrates, K_m and V _max values were determined for guaiacol/H₂O₂ substrate pairs. To this end, the enzyme activities were measured at five different concentrations of guaiacol, while H₂O₂ concentration was constant. This process was repeated for H₂O₂, while guaiacol concentration was constant. K_m and V _max values were calculated from the Lineweaver-Burk graphs, whose results were obtained by the above experiments (Figs. 5a and 5b). The enzyme had K_m values of 17.1 and 3.8 mM for guaiacol/H₂O₂ substrate pattern, respectively. On the other hand, the enzyme had V _max values of 15,500 and 15,889 EU/mL.min for each substrate, respectively (Table 2).

CONCLUSION

Peroxidase from sweet gourd was purified and characterized. This study showed that peroxidase can be purified from sweet gourd. It is the initial reporting of the characterization of sweet gourd POD. Thermal stability of the enzyme was observed to be low. The present study has also revealed that this enzyme has relatively high activity under neutral conditions. As the result of the studies done for determining the stable pH of the enzyme, it is seen that the enzyme is very durable especially under the basic conditions. According to the results, the buffer salt concentration slightly affects POD activity. H₂O₂ was the constant substrate for POD, guaiacol was used as the changeable substrate at the kinetic studies, and it is observed that POD has relatively high V _max and low K_M for this substrate. Finally, sweet gourd can be used as one of the potential POD sources.