Purification, characterization, and inhibition sensitivity of peroxidase from wheat (Triticum aestivum ssp. vulgare)

ABSTRACT

The purification and characterization of peroxidase is currently growing interests since peroxidases have implications in various industrial and biochemical applications. In this study, wheat peroxidase was purified using (NH₄)₂SO₄ precipitation, CM-Sephadex cation exchange, and gel filtration chromatographies. Enzyme purity and molecular mass were checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Enzyme kinetics was studied using two substrates: guaiacol and hydrogen peroxide (H₂O₂). K_m and V_max values were calculated from Lineweaver-Burk graph for each substrate. Wheat peroxidase activity has been enhanced 284-fold. Wheat peroxidase had molecular mass of 38.8 kDa. K_m values for guaiacol and H₂O₂ were found as 2.467 mM and 7.307 mM, respectively. The pH and temperature optima were 5.5 and 40.0°C, respectively. Also, the enzyme was highly inhibited by citric acid and Cetyltrimethylammonium bromide.

KEYWORDS:

• Wheat peroxidase
• Enzyme purification
• Enzyme characterization
• Enzyme inhibition

Introduction

Peroxidase (POD; E.C. 1.11.1.7) is a ubiquitous heme containing plant enzyme, which catalyses the oxidation of various organic and inorganic substrates using H₂O₂ as an electron acceptor.^[1,2] Hydrogen peroxide has important roles as a signaling molecule in the regulation of a wide variety of biological processes.^[3,4] The compound is a major factor implicated in the free-radical theory of aging, based on how readily H₂O₂ can decompose into a hydroxyl and superoxide radicals. Both by-products of cellular metabolism can react with ambient water to form H₂O₂.^[5–7] These hydroxyl radicals in turn readily react with and damage vital cellular components, especially those of the mitochondria.^[8,9] POD is usually located in the membrane and plays crucial role in growing and development as well as construction, rigidification, and eventual lignification of cell walls in the plant.^[10] It is one of the enzymes that has been comprehensively studied and still promotes the new scientific research across the world, because of its commercial use in medical diagnostic kits.^[11] Although new information arises, there are still unanswered questions regarding the function and physiological importance of POD from different plant species.

POD is classified into three families based on amino acid homology and metal-binding capacity.^[12] The first class includes intracellular plant and prokaryotic peroxisomes, such as ascorbate POD or cytochrome cPOD. The second class covers extracellular fungal PODs such as Mn-POD and lignin-degrading POD. The third class of POD is secreted in higher plants and has multiple functions, such as in auxin metabolism, lignin, and suberin formation.^[12,13] Depending on the isoelectric points from acidic to basic medium, different soluble and membrane bound isoenzymes of POD could be found in fruits and vegetables.^[14] Also, one of the most studied POD classes is lactoPOD, which catalyses the oxidation of a number of inorganic and organic substrates by H₂O₂.^[15–18]

Since plant POD has wide range of substrate specificity, it is been used in many areas.^[19] The areas that POD used are mainly immune assays, bioremediation, and synthesis of chemical compounds. Manganese POD has been found to be important in the degradation processes of lignin and other aromatic pollutants, which means that it might have great potential in bioremediation.^[20] The other field that PODs used is biotransformation of various drugs and chemicals in pharmaceutical industry. The preparation of enantiomerically pure compounds is critical for pharmaceuticals and PODs are capable to produce optically active molecules that are environmentally acceptable.^[21] There are number of studies which have been conducted to understand the structural stability of POD due to its wide application in industry. The discovery of PODs from different sources with higher stability is important to promote the usage of it in new analytical methods and industry.^[22]

Purification techniques for POD are well-documented. The methods usually require ammonium sulphate ([NH₄]₂SO₄) fractionation of the sample extract, followed by ion exchange and gel filtration chromatography. There is also one recent study showed that rice bran POD could be purified using expanded-bed adsorption chromatography.^[23] Purification of POD was reported for cauliflower,^[24] Turkish black radish,^[25] avocado,^[26] tea leaves,^[27] and turnip roots,^[28] but little is available about wheat POD.^[29] In this article, the enzymatic properties of wheat POD (wPOD) have been studied. POD was purified using (NH₄)₂SO₄ precipitation, CM-Sephadex ion exchange, and gel filtration chromatographies. The purity of the enzyme was monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Optimum pH, pH stability, optimum temperature, thermal stability, enzyme kinetics, and inhibition of POD by different inhibitors were also determined.

Materials and methods

Plant materials

Locally grown mature wheat spikes were harvested in Erzincan province of Turkey. The wheat species were diagnosed as Triticum aestivum ssp. vulgare by Professor Dr. Ali Kandemir, Erzincan University, Erzincan, Turkey. Wheat spikes were frozen in liquid nitrogen, ground to a fine powder with pestle in a mortar chilled in liquid nitrogen and stored at –20°C until the enzyme extraction was carried out.

Ammonium sulphate precipitation and dialysis

Wheat powder (20 g) was suspended in phosphate buffer (pH: 7.5, 0.1 M). The homogenate was incubated for 30 min at 20°C. Tissue debris was removed by filtration and centrifuged at 8000 × g for 30 min at 4°C. The pellet was discarded and supernatant was collected. The supernatant fractionated by adding solid (NH₄)₂SO₄ for 0–10, 10–20, 20–30, 30–40, 40–50, 50–60, 60–70, and 70–80% intervals and precipitated in an ice bath. The extracts were collected by centrifugation at 12,000 × g for 45 min. The precipitate was suspended in about 2 mL phosphate buffers (pH 7.0, 0.1 M) and the concentrated sample with maximum specific activity was selected for dialysis. The dialysis of the suspension was performed against phosphate buffer (0.1 M, pH 7.0) for 24 h containing 1 mM NaCl.

Preparation of CM-Sephadex A-50 ion exchange chromatography material

After removal of salt by dialysis, the solution was applied to CM-Sephadex A50 ion exchange chromatography at 4°C, pre-equilibrated with Tris-HCl (0.1 M, pH: 9.0). The column was washed with 250 mL of the phosphate buffer and the retained material was eluted using a continuous linear gradient of NaCl (1.0 M), with the same buffer at a flow rate of 15 mL h⁻¹. The fractions were collected as 3 mL tube⁻¹. The collected fractions during elution were immediately analyzed for POD activity and their absorbance measured at 280 nm. Active fractions from the ion exchange chromatography were pooled and stored at 4°C.

Gel filtration chromatography

Active fractions from the ion exchange chromatography were collected and applied on a Sephadex G-25 equilibrated with 6 mM phosphate buffer (pH: 7.0) at 4°C.^[30] POD was eluted with a linear 0–100 mM NaCl gradient at a flow rate of 15 mL/hour. The fractions of 3 mL were collected and followed by measuring absorbance at 280 nm for qualitative protein determination and 470 nm for POD activity determination. The fractions showing POD activity were pooled and the sample then was applied to gel electrophoresis.

Protein electrophoresis

In order to determine the molecular weight (MW) and purified wPOD, the pooled enzyme from gel filtration chromatography was applied to SDS-PAGE.^[31–33] Discontinuous electrophoresis was conducted by using 12.5% separating gel and 4% stacking gel at constant current of 80 volt per gel for 3 h followed by 110 volt reached the bottom of the plate. The gel was stained in Coomassie Blue.^[34]

POD activity

POD activity was determined by measurement of the absorbance at 470 nm using guaiacol and H₂O₂ as substrate.^[35] Briefly, 1 mL of 45 mM guaiacol and 1 mL of 22.5 mM H₂O₂ in 0.1 M phosphate buffer (pH: 7.0) at 4°C and the reaction was started by adding 50 μL of POD solution.

Determination of K_m and V_max

K_m and V_max for wPOD were determined by incubating 50 μL of purified wPOD with different concentrations of guaiacol and a fixed saturated concentration of H₂O₂ from Lineweaver-Burk graph.^[36,37] When guaiacol is used as a substrate the reaction mixture contained 1 mL of different concentrations of guaiacol, 1 mL of H₂O₂ (22.5 mM), 950 µL of phosphate buffer (0.1 mM) and 50 μL of wPOD enzyme. The kinetic data were analyzed by the Lineweaver-Burk plots. The reaction was performed at room temperature (28 ± 2ºC).^[38]

pH and temperature stability

In order to determine the pH and temperature stability of wPOD, different buffer species were used to achieve optimum pH and temperature conditions. The solutions were incubated with intervals of pH (3.5–4.5 with acetate; 5.0–7.0 with phosphate; 7.5–9.0 with Tris buffer) and temperature (10–70°C). The activity of the enzyme was measured in experimental levels between 0–12 days.

In vitro inhibition assay

The in vitro inhibition of wPOD by different inhibitor was also determined. The purified wPOD was mixed with inhibitor at different concentrations (Table 2) for 3 min at room temperature. The remaining specific activities were measured using guaiacol as substrate.

Table 1. The results of purification of peroxidase from wheat (Triticum aestivum ssp. Vulgare).

Purification steps	Total enzyme activity (EU/mL.min)	Total protein (mg)	Specific activity (EU/mg)	Purification fold
Homogenate	0.022	1.152	0.02	1.0
(NH4)2SO4 precipitation and dialysis	0.903	0.892	1.01	51
Ion exchange chromatography	0.139	0.535	0.26	14
Sephadex-G-25	0.881	0.162	5.43	284

Table 2. The inhibition effects of different chemicals on peroxidase (wPOD) activity from wheat (Triticum aestivum ssp. Vulgare).

Inhibitors	Concentration (mM)	Inhibition (%)
Citric acid	5.0	94
EDTA	20.0	89
CTAB	20.0	95
Sodium azide	5.0	79

Enzyme unit

One unit of wPOD enzyme activity was defined as the amount of enzyme that catalyses the production of 1 mmol of tetraguaiacol per minute.^[24]

Protein assay

Total protein concentration was determined using Bradford method. In this assay, the complex forms between Coomassie Brilliant Blue G-250 agent and proteins in the presence of phosphoric acid, and its absorbance is measured at 595 nm.^[39] Samples were prepared in triplicate and bovine serum albumin was used as standard protein.^[40]

Results and discussion

Purification studies

In this study, wPOD was successfully purified through three steps: ammonium sulphate precipitation, CM-Sephadex cation exchange, and gel filtration chromatography. The specific activity, fold-purification, and the yield (%) for each step are presented in Table 1. For the partial purification of the wPOD enzyme (NH₄)₂SO₄ precipitation was performed. Solid (NH₄)₂SO₄ was added in increasing saturation from 10 to 80 (w/v) into supernatant with continuous string at 0°C for 20 min. After centrifugation, wPOD enzymatic activity of both precipitate and supernatant were measured at each interval. There was almost no wPOD activity at the precipitate with 10% ammonium sulphate saturation; however, it was substantially increased after dissolving the precipitate obtained by 50–60% (NH₄)₂SO₄ saturation (Fig. 1a). This step resulted in removal of significant amount of other protein in the extract with 53-fold purification. In also previous studies (NH₄)₂SO₄ was used in the purification step of POD from Tartary buckwheat shoots.^[41]

Figure 1. Purification of peroxidase (wPOD) purified from wheat (Triticum aestivum ssp. Vulgare). A: Ammonium sulphate precipitation; B: CM-Sephadex anion exchange chromatography; and C: Gel filtration chromatography.

After removal of salt by the dialysis, the sample was applied to directly to an open CM-Sephadex cation exchange column for further purification. The fractions were collected 1.5 M NaCl and 0.1 M phosphate gradient. For each fraction, the amount of protein was measured spectrophotometrically at 280 nm and wPOD enzymatic activity was measured at 470 nm (Fig. 1b). A previous study where wPOD was purified from wheat kernel using cation exchange chromatography showed that wPOD has three different enzyme forms. In addition, it has been shown that using anion exchange chromatography wPOD purified from broccoli had two isoenzymes, one of which being less pure.^[42] In this study, only one peak with wPOD activity was obtained using cation exchange chromatography (Fig. 1b). This elution profile indicated that wPOD has a net cationic surface charge that allowed them to be absorbed onto negatively charged CM-Sephadex column chromatography. The peak values for wPOD activity appeared at between 12–17 fractions. These factions were pooled together and applied to gel filtration chromatography.

Gel filtration chromatography is one of the steps used for purification of POD, for example from wheat bran.^[43] wPOD activity and protein flow during gel filtration chromatography was shown in Fig. 1c. For each fraction, the amount of protein was measured spectrophotometrically at 280 nm and wPOD activity was measured at 470 nm. The elution profile showed only one peak for wPOD activity. The peak for highest enzymatic activity appeared at between 12–17 fractions. The result showed that after ion-exchange chromatography, POD was successfully purified for 284 folds (Table 1). The extraction conditions employed resulted in an increase in specific activity from 0.02 (homogenate) to 5.43 U/mg (Sephadex G-25) via fractional purification of desired enzyme and elimination of contaminating enzymes or proteins. Although a 284-fold increase was achieved for final specific activity of wPOD, the greater specific activity of 8.90 U/mg for cowpea leaves^[44] and 580 U/mg for turnips^[45] using the same techniques.

MW and purity

The active fractions obtained from gel filtration chromatography were pooled and checked for purity by SDS-PAGE. Electrophoretic results of purified wPOD were shown in Fig. 2. One clear protein band appeared for purified wPOD indicating a monomer with MW of between 35 and 50 kDa. The exact MW of purified wPOD was calculated as 38.8 kDa with SDS-PAGE method. There are different results regarding the MW of plant PODs. For example it has been reported that POD has MW of 32 kDa for Acorus calamus,^[46] 33 kDa for rosemary,^[47] 36 kDa in turnip root,^[48] 40 kDa in horseradish, and 43 kDa for Aspergillus terreus.^[49] To our knowledge, the highest MW for plant POD was reported for cabbage leaves with 67 kDa.^[10] The differences in the MW of plant POD could be due to differences in amino acid sequences or different levels of glycosylation.^[50]

Figure 2. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) zymograme of peroxidase (wPOD) purified from wheat (Triticum aestivum ssp. Vulgare). Molecular weight markers (Lane 1), and gel filtration peak (Lane 2–3).

Characterization studies

The active site of POD enzyme is mainly composed of ionic groups (prosthetic groups) and must be kept in a certain conformation so that the enzyme maintains the catalytic activity and substrate binding property. pH is a crucial factor for determining the POD enzyme activity since it changes the ionization state of the amino acids or substrate. The effect of pH on POD activity is shown in Fig. 3a. A pH range of from 3.0 to 9.0 was studied. The activity of the enzyme sharply increased at pH: 4.0 and was stable within the pH range of 4.5–7.0, followed by a decrease at pH 8.0. Similar results were reported for different plant PODs in the literature. For example the optimum pH for maximum activity was 6.0 for rosemary,^[47] 5.5 for a chickpea cultivar,^[51] 4.6 for papaya,^[52] and 5.0 for oil palm.^[53] The loss of activity at lower and higher pH could be due to the loss of heme group, which is found in the active site of POD.^[29]

Figure 3. The effect of different conditions on peroxidase (wPOD) purified from wheat (Triticum aestivum ssp. Vulgare). A: Optimum pH of wPOD; B: The enzyme stability at different pH with time; C: The effect of temperature on wPOD activity; D: Thermal stability of wPOD; and E: The effect of ionic strength on wPOD.

In order to determine pH stability of the enzyme, aliquots of the sample were incubated at different pH varying from 3.0 to 9.0 at different time points (0–7 days; Fig. 3b). At the beginning, the wPOD activity was relatively lower at pH 3.0, 8.0, 9.0 and higher at pH 5.0, 6.0, and 7.0, which is consistent with optimum pH study. However, after 2 days of incubation, the activity of enzyme increased in all pH point except from at pH 9.0. After the second day, all samples at different pH started to show decrease in wPOD activity except from the sample at a pH of 4.0. wPOD activity was maintained up to 7 days at pH 4.0, whereas samples at other pHs indicated decrease in the enzymatic activity on 7th day.

The optimum temperature of wPOD activity was studied at temperatures between 10–70°C (Fig. 3c). The result indicated that wPOD activity was highest at 40°C. After that point, wPOD activity gradually decreased. The same optimum temperature was found for Citrus jambhiri.^[54] Interestingly, optimum temperature of POD purified from leaves of thymbra plant (Thymbra spicata L. var. spicata) is substrate dependent and was found 40°C for ABTS, guaiacaol and o-dianisidine, 50°C for o-phenylenediamine, and 20°C for catechol.^[55]

Thermal stability of wPOD was also studied at different temperature and time points (Fig. 3d). The wPOD activity maintained the highest activity at 40°C at pH 4 for 60 min. wPOD activity increased at 30°C, whereas it decreased at 50 and 60°C over the time. This result also confirmed that POD is stable and shows highest activity at 40°C at 30 min. At all temperatures the enzymatic activity reduced after 40 min, which indicates that the activity measurement should be performed less than 40 min. Previously, it was found that optimum temperature of POD is variable. For example it was 60°C for horseradish POD,^[56] 30°C for Jatropha curcas leaves,^[57] and 30°C for cowpea leaves. It was also found that the optimum temperature was 0–40°C for Pinus pinaster needles,^[58] over 50°C for melon,^[59] and at between 25–40°C for garlic Allium sativum.^[60]

Proteins could be stabilised with salt by controlling the water activity around the enzyme.^[60] Therefore, ionic strength and concentration of the salt affects the protein stability. Based on the cavity theory, it has been suggested that the stability of the proteins in aqueous solutions could change depending on the presence of the salt due to the surface tension increment of water.^[61] The effect of ionic strength on wPOD activity was investigated by measuring wPOD activity in buffers with different ionic strength (Fig. 3e). The wPOD activity was highest when the ionic strength was 0.1 mM, whereas it was lower when the ionic strength is lower or higher than 0.1 mM. A previous study showed that wPOD had maximum activity at 0.3 M of phosphate buffer concentration at pH 6.0 and lower activity at concentrations at below or over 0.3 M.^[47] In addition POD purified from leaves of spinach showed maximum activity when the ionic strength was 0.075 M.^[24] A different study indicated that the optimum ionic strength for POD from sweet gourd was 0.4 M.^[25]

Kinetic studies

POD has a wide range of substrate specificity and it can catalyse oxidation of guaiacol, o-phenylenediamine, o-dianisidine, pyrogallol, and p-aminoantipyrine through a reaction with hydrogen peroxide.^[62] It has been suggested that the reduction potential of active site of the enzyme could be affected by electrostatic field caused by the charged residues of the enzyme.^[63] To study the kinetics of the enzyme action, the oxidation of guaiacol was measured at 470 nm and the K_m was calculated using Lineweaver-Burk plot. The kinetic of the reaction was studied at different concentration of the substrate. The Michaelis-Menten and Lineweaver-Burk plot where guaiacol is used as variable substrate and H₂O₂ is used at saturated concentrations was shown in Fig. 4a. A Lineweaver-Burk plot where H₂O₂ is used as variable substrate and guaiacol is used at saturated concentrations was shown in Fig. 4b.

Figure 4. A: Lineweaver-Burk plots for both substrate of guaiacol; and B: H₂O₂ for peroxidase (wPOD) purified from wheat (Triticum aestivum ssp. Vulgare).

Lower K_m values mean that the enzyme has a higher affinity to substrate.^[12] The calculated K_m values for wPOD were 6.6 and 7.3 mM for guaiacol and H₂O₂, respectively. Previously, K_m values for POD were found 0.296 mM for avocado,^[26] 9.5 mM for garlic Allium sativum,^[64] 0.026 mM for garlic,^[65] and 0.22 mM for tomato cell suspension cultures.^[66] In addition cationic and anionic isoPOD purified from cowpea leaves have K_m values of 0.8 and 4.8, respectively.^[44]

Inhibition effect of various chemicals on enzyme activity

PODs are oxidoreductases found in a variety of microorganisms as well as plants. No doubt that PODs have many applications for commercial use due to having a wide range of substrate specifity. However, there are some limitations regarding their use, which include inactivation of POD by peroxides, the low water solubility of the substrates of interest and requirement of relatively high temperatures limiting their applications in low temperatures. Therefore, the discovery of new POD enzymes having different characterizations is important to supply a new source for potential applications. In this study, we report purification, characterization, and identification of wPOD from wheat (Triticum aestivum ssp. vulgare). Optimal conditions for the best activity were determined. K_m and V_max were calculated for both guaiacol and H₂O₂. The effects of different inhibitors were also investigated. The information provided here may be useful for purification of wPOD from different species and for potential applications where POD is used.

Conclusion

Due to having a wide range of biochemical reaction, POD is valuable enzyme in industry. The discovery of new POD enzymes having different characterizations is important to supply a new source for potential applications. Optimal conditions for the best activity of wPOD were determined. K_m and V_max were calculated for both guaiacol and H₂O₂. The effects of different inhibitors were also investigated. The information provided here may be useful for purification of POD enzyme from different species and for potential application that POD is used.