Abstract: In present study, it is aimed to purify peroxidase (EC 1.11.1.7; donor: Hydrogen peroxide oxidoreductase) from leaves of spinach and determine its some biochemical properties. Peroxidase is an oxidoreductase enzyme produced by a number of organisms. Spinach (Spinacia oleracea L.) is an edible flowering plant in the family of Amaranthaceae and is heavily consumed all over the world. Peroxidase enzyme was purified from Leaves of Spinach (Spinacia oleracea L.) by ammonium sulphate precipitation and CM-Sephadex ion-exchange chromatography. Km (Michaelis constant) and Vmax (The maximum velocity of an enzymatic reaction) values were calculated from the Lineweaver-Burk graph for catechol and guaiacol substrates and the substrate specificity of the enzyme was investigated. The results have shown that guaiacol is a better substrate from catechol for this enzyme. Furthermore, for the guaiacol substrate, optimum pH, optimum temperature, optimum ionic strength, stable pH, conditions were determined.
INTRODUCTION
Peroxidases (E.C.1.11.1.7) (POD) are haem proteins and contain iron (III) protoporphyrin IX (ferriprotoporphyrin IX) as the prosthetic group. These are a group of oxidoreductases that catalyse the reduction of peroxides, such as hydrogen peroxide and the oxidation of a variety of organic and inorganic compounds (Hamid and Rehman, 2009). POD is a major system for the enzymatic removal of H2O2 and peroxidative damage of cell walls is controlled by the potency of antioxidative peroxidase enzyme system (Velikova et al., 2000). POD is found in many plant-based foods and plays roles in food quality, including deterioration of colour and flavour (Ashie et al., 1996). Peroxidase activity has been identified in plants, microorganisms and animals, where peroxidases play important roles (Hamid and Rehman, 2009). In plants POD participate in the lignification process (Wakamatsu and Takahama, 1993) and in the mechanism of defence in physically damaged or infected tissues (Biles and Martyn, 1993). On the other hand, POD can contribute to adverse changes in the flavour and colour of fruits and vegetable (Dogan et al., 2007). POD is involved in plant hormone regulation, senescence of plants and defence indoleacetic acid degradation during maturation (Brooks, 1986). In addition, this enzyme 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 (Kwak et al., 1996). Also economically, POD is important because it is well-suited for the preparation of enzyme-conjugated antibodies (EL İLSA), due to its ability to yield chromogenic products at low concentrations and its relatively good stability (Duarte-Vazquez et al., 2001).
In present study, it is aimed to purify POD from leaves of spinach as new source and identify its biochemical properties such as substrate specificity, optimum pH, optimum temperature, optimum ionic strength and stable pH conditions.
MATERIALS AND METHODS
Plant materials and chemicals: Fresh leaves of spinach were obtained from a local market in Erzincan, Turkey. Then, it was washed, peeled, broken up, packed in polyethylene bags, and stored at -83°C until the enzyme extraction (Gulcin et al., 2004a). Also, all chemicals of analytical grade were obtained from Merck.
Preparation of plant extract: Thirty gram leaves of spinach 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 75 mL of phosphate buffer (pH 7.0, 0.1 M) and subsequently was centrifuged at 10.000 xg for 90 min at 4°C. The buffer composition was as follows: 0.1 M phosphate buffer; PVP (0.05%); pH 7.0 (Koksal and Gulcin, 2008). The pellet was discarded. The crude extract was subjected to ammonium sulphate fractionation and the precipitate in the 50-90% saturation range was collected by centrifugation for 90 min at 10.000 g. The precipitate was suspended in about 2 mL phosphate buffer (pH 7.0, 0.1 M) and dialyzed for 8 h at 4°C against 10 mM 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: The 3.5 g dried CM-Sephadex A-50 (Sigma) was dissolved in 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 with 100 mL NaOH (0.5 N) and was allowed to stand for 1 h. Afterwards, the supernatant was decanted and the exchanger was washed with distilled water until the effluent had 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 (3x30 cm) washed and equilibrated with the same buffer. The flow rates for washing and equilibration were adjusted by peristaltic pump as 15 mL h-1 (Robyt and White, 1987).
Purification of peroxidase by cation exchange chromatography: Briefly, 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 dialyzed enzyme extract was loaded onto column and washed with equilibrating buffer. Retained proteins were eluted with a gradient of (250 mL) 0-1 M NaCl in 10 mM phosphate buffer at a flow rate of 15 mL h-1 by using a gradient mixer apparatus (Pharmacia Fine Chemicals). Fractions of 5 mL were collected and activity and absorbance of each were separately measured at 470 and 280 nm, respectively (Fujita et al., 1997).
Enzyme activity assay: The POD activity in the leaves of spinach sample was measured using catechol and guaiacol as the substrate. Temperature was controlled using a circulating water bath with a heater/cooler (Grant LTD 6G -20 to 100°C, England). The changes in absorbance were read for 3 min using a double beam UV-VIS spectrophotometer (CHEBIOS s.r.l.). 470 and 295 nm were used as wave length for guaiacol and catechol substrates, respectively. The final mixture contained 25 μL enzyme sample, 1 mL 22.5 mM H2O2, 1 mL 45 mM guaiacol or catechol and the 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 was monitored for 3 min at 25°C (Fujita et al., 1997; Gulcin et al., 2005).
The determination of protein amount: The determination of qualitative protein amount was measured at spectrophotometrically 280 nm (Soyut and Beydemir, 2008). During the purification process, the determination of quantitative protein amount was carried out spectrophotometrically at 595 nm by using the method of Bradford (1976). Bovine serum albumin as a standard protein (Gulcin et al., 2004b; Hisar et al., 2005; Coban et al., 2008).
Some biochemical properties
The effect of 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. The process was carried
out by a circulatory water bath in a temperature range between 0 and 90°C.
Optimum pH: 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 (Koksal and Gulcin, 2008).
Stable pH: POD activity was observed for 10 days for determine the stable pH value of the enzyme. The process was carried out using three different buffers with pH range between pH 3 and 9 at 0.5 pH intervals.
Effect of ionic strength: The effect of ionic strength on the enzyme activity was studied using different concentrations of buffers (0.025-0.25 M). Studies were carried out at predetermined optimal pH (Koksal and Gulcin, 2008).
Kinetic studies: So as to determine Km and Vmax values of guaiacol and catechol substrates, the enzyme activities were measured using five different concentrations of the substrates. Km and Vmax values were calculated from the Lineweaver-Burk graphs, whose results were obtained by the above experiments. Km and Vmax values were calculated for peroxidase reactions with each of the two substrates, using the Lineweaver-Burk transformation of the Michaelis-Menten equation (Lineweaver and Burk, 1934). The Vmax and Km ratio is called as the catalytic power and the value of this ratio determines the more effective substrate (Dogan et al., 2007).
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 M NaCl in 100 mM phosphate buffer (pH 8.0) at flow rate of 10 mL h-1.
| Table 1: | Summary of the purification stages of spinach leaves (Spinacia oleracea) peroxidase |
| Fig. 1: | Cation exchange chromatography of POD from Leaves of Spinach (Spinacia oleracea 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 |
| Fig. 2: | Changes in the peroxidase activity from Leaves of Spinach (Spinacia oleracea L.) at different temperatures |
| Fig. 3: | Changes in the peroxidase activity from Leaves of Spinach (Spinacia oleracea L.) at different pH |
| Fig. 4: | The effect of incubation period on the peroxidase activity from Leaves of Spinach (Spinacia oleracea L.) at different pH values. A: pH 4.5-6.0 and b: pH 6.5-8.0. |
Eluates were collected as 5 mL fractions and the activity and absorbance of each were separately measured at 470 and 280 nm (Beydemir and Gulcin, 2004; Aras-Hisar et al., 2004), respectively (Fig. 1). As a result of this process, POD was purified in 22.6 fold with 4.2% yield (Table 1).
Characterization studies: In order to determine the optimum temperature values of the enzyme, POD activity was measured at different temperatures in a range between 0 and 90°C. As shown in Fig. 2, the optimum temperature of peroxidase enzyme was determined to be 60°C. It was observed that the enzyme activity increased to 60°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 (Beydemir et al., 2003). As can be shown in Fig. 3, the optimal pH of peroxidase from leaves of spinach was determined to be 5.2 using 0.1 M sodium phosphate buffers.
| Fig. 5: | Kinetic behaviour of the two substrate reactions for Leaves of Spinach (Spinacia oleracea L.) POD (a) Plot of guaiacol substrate. (b) Plot of catechol substrate |
| Table 2: | Optimum pH, optimum temperature, optimum ionic strength and stable pH values of peroxidase from spinach leaves (Spinacia oleracea) |
| Table 3: | Vmax and Km values of peroxidase from sweet gourd (Cucurbita Moschata L.) |
To assign the stable pH value of the enzyme, enzyme activity was monitored in three different buffers for 10 days (Koksal and Gulcin, 2008) and it was demonstrated that POD was more stable in acidic conditions at the end of this incubation period. The results were shown in Fig. 4a, b.
The results of the ionic strength study revealed that the salt concentration affects POD activity. POD activity was measured at 75 mM of buffer concentration as maximum value.
Kinetic studies: Km and Vmax values were determined for guaiacol and catechol substrates. To this end, the enzyme activities were measured at five different concentrations of substrates while constant concentration of H2O2 (Dogan et al., 2007). Km and Vmax values were calculated from the Lineweaver-Burk graphs, of which results were obtained by the above experiments (Fig. 5.a, b). The enzyme had Km values of 17.35 mM and 23.15 mM for guaiacol and catechol substrates, respectively. On the other hand, the enzyme had Vmax values of 1234 and 645 Eu mL-1. min for each substrate, respectively (Table 2). The value of Vmax and Km ratio was higher for guaiacol and therefore guaiacol was determined as a better substrate than catechol.
CONCLUSION
Briefly, peroxidase enzyme (EC 1.11.1.7; donor: Hydrogen peroxide oxidoreductase) from spinach leaves (Spinacia oleracea L.) was extracted, purified and characterized by determination of its some biochemical properties. It was showed that peroxidase could be partially purified from spinach leaves at only two steps, ammonium sulphate precipitation and CM-Sephadex ion-exchange chromatography. As the result of the studies for determining the stable pH of the enzyme, it was seen that the enzyme was very durable especially under the acidic conditions. According to the results, the buffer salt concentration slightly affected POD activity. Optimal active pH was at 5.2 and optimal temperature at 60°C for POD of spinach leaves. According to this result, the enzyme has higher activity than neutral and alkaline conditions at acidic conditions and the enzyme can protect and increase its activity up to 60 degrees. H2O2 was constant substrate for POD, guaiacol and catechol was used as changeable substrate at the kinetic studies and the results have shown that guaiacol is a better substrate than catechol for this enzyme, that is, POD has higher substrate specificity for guaiacol substrate. Purification of the POD and its determination of the some biochemical properties were important for understanding the enzyme in spinach leaves. Also, this study is the first report on spinach leaves POD.
ACKNOWLEDGMENT
I would like to thank to my dear friend and colleague Hamit Mermerkaya for improving the English of the paper.