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Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties
Accepted Manuscript Title: Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties Authors: Abdelbasset Chafik, Abdelkhalid Essamadi, Safinur Yildirim C ¸ elik, Ahmet Mavi PII: DOI: Reference:
S1570-0232(17)31251-5 https://doi.org/10.1016/j.jchromb.2017.10.052 CHROMB 20882
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
19-7-2017 17-10-2017 26-10-2017
Please cite this article as: Abdelbasset Chafik, Abdelkhalid Essamadi, Safinur Yildirim C ¸ elik, Ahmet Mavi, Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties, Journal of Chromatography B https://doi.org/10.1016/j.jchromb.2017.10.052 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties Abdelbasset Chafika*, Abdelkhalid Essamadia, Safinur Yildirim Çelikb, Ahmet Mavic a
Laboratory of Biochemistry and Neuroscience, Team of Applied Biochemistry and
Toxicology, Faculty of Science and Technology, University Hassan First, 577, Settat, Morocco b
c
College of Education, Bayburt University, 69000, Bayburt, Turkey
Chemistry Education, Kazim Karabekir Education Faculty, Atatürk University, 25240, Erzurum, Turkey
*The author to whom correspondence should be addressed Abdelbasset Chafik Laboratory of Biochemistry and Neuroscience, Team of Applied Biochemistry and Toxicology, Faculty of Science and Technology, University Hassan First, 577, Settat, Morocco E-mail: [email protected] Phone: +212 762560876
Highlights
The camel is often subjected to intense environmental stress in the desert. Catalase plays a key role in protecting cells against oxidative stress. Catalase from camel liver was purified by zinc chelate affinity chromatography. The properties of purified catalase were different comparing to those of mammals. The catalase could play an important role in the camel's adaptation to its milieu.
Abstract: Climate change and increasing temperatures are global concerns. Camel (Camelus dromedarius) lives most of its life under high environmental stress in the desert and represent ideal model for studying desert adaptation among mammals. Catalase plays a key role in protecting cells against oxidative stress. For the first time, catalase from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution, a better separation was obtained. A purification fold of 201.81 with 1.17% yield and a high 1
specific activity of 1132539.37 U/mg were obtained. The native enzyme had a molecular weight of 268 kDa and was composed of four subunits of equal size (65 kDa). The enzyme showed optimal activity at a temperature of 45°C and pH 7.2. Thiol reagents, β-Mercaptoethanol and D,L-Dithiothreitol, inhibited the enzyme activity. The enzyme was inhibited by Al3+, Cd2+ and Mg2+, whereas Ca2+, Co2+ and Ni2+ stimulated the catalase activity. Reduced glutathione has no effect on catalase activity. The Km and Vmax of the enzyme for hydrogen peroxide were 37.31 mM and 6185157 U/mg, respectively. Sodium azide inhibited the enzyme noncompetitively with Ki value of 14.43 µM, the IC50 was found to be 16.71 µM. The properties of camel catalase were different comparing to those of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of reduced glutathione from hydrogen peroxide oxidation and higher affinity for hydrogen peroxide and sodium azide, these could be explained by the fact that camel is able to live in the intense environmental stress in the desert.
Abbreviations: ROS: Reactive oxygen species; CAT: Catalase; H2O2: Hydrogen peroxide; MCAC: Metal chelate affinity chromatography; GSH: Reduced glutathione; DTNB: 5,5′Dithiobis(2-nitrobenzoic acid); β-ME: β-Mercaptoethanol; DTT: D,L-Dithiothreitol; PMSF: Phenylmethylsulfonylfluoride; EDTA: Ethylenediaminetetraacetate; NaN3: Sodium azide.
Keywords: Camel; Camelus dromedarius; Desert; Catalase; Affinity chromatography; pH gradient elution.Introduction The one-humped camel (Camelus dromedarius), is among the mammals domesticated by humans for their needs in the desert, as it is a multipurpose animal used for production, leisure, transport, agricultural work or treatment of diseases [1, 2]. It is well known that camel has some biochemical, anatomical and physiological peculiarities due to their adaptation to desert life and differ it from other mammalian species [3, 4], although much less is known about physiological and biochemical studies of enzymes involved in the metabolism of reactive oxygen species (ROS) and oxidative stress in this animal, especially with the climatic changes and increasing temperatures [5]. The camel is continuously exposed to heat, solar radiation, and lack of water and food [6, 7]. This specific ecosystem could result in a disturbance in the balance between the production 2
of ROS and antioxidant defenses, and therefore causing damage of important biomolecules such as DNA, proteins and enzymes [8]. Generally, living organisms are equipped with both enzymatic and non-enzymatic antioxidant defense mechanisms to protect themselves against ROS [9]. Catalase (CAT, EC 1.11.1.6) is one of the principle antioxidant enzymes, which plays a key role in protecting cells from oxidative damage. It catalyzes the conversion of hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2) [10]. CAT has been purified and characterized from a large number of microorganisms [11–15] and plants [16–19]. In mammalian species, the enzyme has been purified and characterized from human [20–23], bovine [24, 25], porcine [26, 27], goat [28, 29], rat [30, 31], dog [32, 33], mouse [34], horse [35] and sheep [36]. However, information on CAT from camel is limited. Until now only one report has appeared on the partial purification and characterization of this enzyme from camel liver [37]. On the other hand, numerous purification procedures of CAT are frequently used ion exchange chromatography and gel filtration chromatography. Purification of CAT from mammalians [21, 25, 34], plants [16] and microorganisms [12, 13, 15] using metal chelate affinity chromatography (MCAC) is limited. Step-wise increase in the concentration of imidazole [12, 13, 15, 16, 25, 34] or linear gradient of histidine [21] were used to elute the adsorbed CAT from MCAC. The method of purification of CAT by MCAC using pH gradient elution has not been reported. This paper describes for the first time the purification of CAT from camel liver by MCAC using pH gradient elution, and compares some of its properties with those of other CATs. The study of this protein biomarker will contribute to understanding some of the mechanisms involved in the resistance to stress induced by the life in the desert. 1. Materials and methods 1.1. Chemicals DEAE-Sepharose, imminodiacetic acid immobilized Sepharose 6B, Sephacryl S-200, gel filtration markers kit for protein molecular weights 12–200 kDa, 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB), reduced glutathione (GSH), phenylmethylsulfonylfluoride (PMSF) and D,LDithiothreitol (DTT) were purchased from Sigma Aldrich. PageRuler™ Plus Prestained Protein Ladder 10–250 kDa were purchased from ThermoFisher Scientific. All other chemicals were of analytical grade. 1.2. Purification of CAT 3
1.2.1. Ethanol-Chloroform treatment Fresh camel liver was obtained from municipal slaughterhouse of Casablanca–Morocco. Approximately 73 g of liver was homogenized in 2 volumes of cold potassium phosphate buffer (20 mM, pH 7.0) using a Waring blender. The mixture was then centrifuged at 3500 × g for 20 min at 4°C. The hemoglobin was precipitated by chloroform-ethanol treatment according to Tsuchihashi [38]. With stirring, 0.25 volume of ethanol and 0.15 volume of chloroform, precooled in a freezer (-24°C), were added rapidly to the lysate. After 15 min, the mixture was centrifuged at 3500 × g for 60 min at 4°C. Thereafter, the supernatant was dialyzed against four changes of 20 mM potassium phosphate buffer pH 7.0, 20 volumes each, over a period of eight hours at 4°C. 1.2.2. DEAE-Sepharose ion exchange chromatography The precipitate which formed during dialysis was removed by centrifugation and the supernatant was loaded on a DEAE-Sepharose column (2.5 × 10 cm) pre-equilibrated with the same buffer as that used for dialysis. The adsorbed proteins were eluted at room temperature with a linear gradient of 0 to 0.5 M KCl prepared in the equilibration buffer at a flow rate of 60 ml/h. Fractions of 2 ml were collected and the fractions containing CAT activity were subjected to zinc chelate affinity chromatography. 1.2.3. Zinc chelate affinity chromatography An affinity column (1.6 × 9 cm) was packed with imminodiacetic acid immobilized Sepharose 6B and activated with 20 mM ZnSO4.7H2O solution. A second column (1.4 × 4 cm) was packed with the same gel, which did not receive the zinc solution, and connected to the bottom of the column containing the metal-activated gel. The second column served as a guard to adsorb leakage of zinc ions from the first column. The sample was applied to the first column equilibrated with 20 mM potassium phosphate buffer pH 7.0 containing 0.1 M NaCl. After washing with the same buffer, CAT was eluted at room temperature with a linear gradient of pH from 20 mM potassium phosphate buffer pH 7.0 to 20 mM citrate buffer pH 5.0, both buffers containing 0.1 M NaCl. A constant flow-rate of 60 ml/h was maintained throughout the procedure. The activity of CAT was determined in all the eluted fractions of 1 ml and activitycontaining fractions were collected and stored at -24°C until use. 1.2.4. Homogeneity of the purified enzyme 4
Homogeneity of the purified enzyme was confirmed by electrophoretic analysis using a vertical slab gel apparatus. Native gel electrophoresis was carried out with 7.5% PAGE according to Davis [39]. SDS-PAGE was performed with 12% polyacrylamide gel according to Laemmli [40]. The proteins were stained with coomassie brilliant blue G-250. 1.3. Characterization of CAT 1.3.1. Molecular weight determination The native molecular weight of the purified enzyme was estimated using Sephacryl S-200 (1.75 × 37 cm) gel filtration column. The column was equilibrated with 50 mM Tris-HCl (pH 7.5) containing 0.1 M KCl and calibrated with the following standard proteins: Cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), albumin (66 kDa), alcohol dehydrogenase (150 kDa) and β-Amylase (200 kDa). CAT was eluted with the above-mentioned buffer at a flow rate of 0.5 ml/min and the enzyme activity was determined in the collected fractions of 1 ml. The molecular weight of the subunits of the enzyme was estimated by SDS-PAGE according to the method of Weber and Osborn [41], using 12% polyacrylamide gel with a current of 50 mA for approximately 4 h. Molecular weight marker proteins used for SDS-PAGE were from commercial grade with molecular mass ranging from 10 to 250 kDa. Protein bands were visualized in gel by staining with 0.3% coomassie brilliant blue G-250. 1.3.2. Optimum temperature and pH The optimum temperature of the enzymatic reaction of purified enzyme was determined over a temperature range from 20 to 80°C. To determine optimum pH for enzymatic reaction, activity of purified enzyme was measured using two different buffer systems in two different pH ranges. A 0.05 M phosphate buffer was used for pH 6.0–8.0 intervals and a 0.05 M Tris-HCl buffer was used for pH 8.6–9.0 intervals. 1.3.3. Effect of inhibitors, metal ions and reduced glutathione The effect of inhibitors, metal ions and reduced glutathione (GSH) on the activity of purified enzyme were investigated. β-Mercaptoethanol (β-ME), ethylenediaminetetraacetic acid (EDTA), D,L-Dithiothreitol (DTT), phenylmethylsulfonylfluoride (PMSF) and sodium dodecyl sulfate (SDS) were tested at a final working concentration of 2 and 5 mM. The purified enzyme was incubated with a final working concentration of 1, 2 and 5 mM of salts of metal ions (chlorides of Al3+, Ca2+, Cd2+, Co2+, Mg2+ and Ni2+) to tested the effect of these metal ions 5
on the activity of purified enzyme. Since non-enzymatic reaction between GSH and H2O2 occurred [42, 43], the effect of GSH on the non-enzymatic reaction and on CAT activity was studied by varying the concentration of GSH from 0.5 to 5.0 mM. The residual GSH was determined according to the procedure of Sedlak and Lindsay [44] based on Ellman's reagent [45], with slight modifications. After 1 min, according to the assay system of CAT activity, 250 µl of 0.04% 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) prepared in 0.4 M phosphate buffer pH 7.0 was added immediately in the cuvette. The optical density measured at 412 nm after 2 min, is directly proportional to GSH concentration in the sample. The GSH concentration in sample was determined by reference to the standard curve of GSH. 1.3.4. Kinetic parameters Purified enzyme was used to calculate Km and Vmax of CAT for H2O2, and Ki, IC50 and Hill slope of CAT for sodium azide (NaN3). CAT activity was measured as described in assay system. The Km, Vmax and Ki were determined by varying concentrations of NaN3, from 0 to 80 µM, and keeping H2O2 concentration constant at 10, 20 and 30 mM; experiments to determine IC50 and Hill slope were carried out with varying concentrations of NaN3, from 0 to 500 µM, and keeping H2O2 concentration constant at 10 mM. Data were analyzed with nonlinear regression using GraphPad Prism software version 7. 1.3.5. Assay of CAT activity CAT activity was assayed according to the method of Aebi [46]. The assay was performed in a reaction mixture containing 10 mM H2O2 in 50 mM potassium phosphate buffer pH 7.0, and the reaction was started by addition of enzyme solution. CAT activity was determined spectrophotometrically by monitoring the decrease in H2O2 absorption at 240 nm for 1 min. One unit of CAT activity was defined as the calculated consumption of 1 µmol of H2O2 per min at 25°C. The molar extinction coefficient for H2O2 is 43.6 M-1 cm-1. The experiments were done in triplicate. Staining of CAT activity on native PAGE was performed according to the procedure of Woodbury et al. [47]. After electrophoresis, the gel was soaked for 10 min in 0.01% of H2O2. The gel was then stained with a reaction mixture containing 1% potassium ferricyanide and 1% ferric chloride. Achromatic bands of CAT appeared against a green background. 1.3.6. Protein determination 6
Protein concentrations were measured by the dye binding assay method of Bradford [48] using bovine serum albumin as a standard. 2. Results and discussion 2.1. Purification and homogeneity After precipitation of hemoglobin by ethanol-chloroform treatment, CAT from camel liver was purified by two steps of column chromatography using DEAE-Sepharose and zinc chelate affinity chromatography. CAT purified from mammalians [25, 34], plants [16] and microorganisms [12, 13, 15] using MCAC was generally eluted by step-wise using a specific eluent such as imidazole. For the first time, CAT from camel liver was purified by zinc chelate affinity chromatography using pH gradient elution. The CAT was eluted with a linear gradient of pH using 20 mM potassium phosphate buffer pH 7.0 and 20 mM citrate buffer pH 5.0, both buffers contain 0.1 M NaCl. As shown in Figure 1A, CAT was eluted between pH 6.13 and 5.28. A better separation was obtained using pH gradient elution, which removed a small amount of the contaminating proteins. In the same manner, contaminating proteins were separated using linear gradient of histidine [21]. The purification profile of CAT from camel liver is given in Table 1. The specific activity of the final preparation was 1132539.37 U/mg protein, which represents 201.81-fold purification with a 1.17% yield. The specific activity of the purified camel liver CAT was higher than that of purified CAT from different organs of human [20–23, 49], liver and milk of bovine [24, 25], porcine kidney [26], goat lung [28], liver and erythrocytes of dog [32, 33], mouse liver [34], sheep erythrocytes [36] and murine erythrocytes [49]. The purification fold of the purified CAT by MCAC was reported to be 4.2-fold in bovine liver [25], 12 and 44-fold in mouse liver [34], 38.7 and 161.8-fold in bacteria [12, 15], 23.3 and 106.8-fold in plant [16]. The purification fold achieved in this study (201.81-fold) was higher than that in reported studies. The 565-fold purification was reported in human erythrocytes CAT purified by MCAC [21]. CAT from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution. The final enzyme preparation was subjected to PAGE and SDSPAGE. The purified enzyme gave a single protein band when the gel was stained for CAT activity (Figure 1B) and for protein with coomassie brilliant blue G-250 (Figure 1C). Also, the purified enzyme was eluted from Sephacryl S-200 column as a single peak of enzyme activity (Figure 1D). 7
2.2. Characterization and some properties of purified CAT 2.2.1. Molecular weight estimation The native molecular weight of CAT eluted from Sephacryl S-200 column was found to be 267827 Da (Figure 2A). The subunit molecular weight of the enzyme was estimated to be 64597 Da from SDS-PAGE, as shown in Figure 2B. These results suggested that the camel liver CAT was tetramer composed of four identical subunits. The molecular weight of camel liver CAT was relatively higher than that of purified CAT, containing four identical subunits, from white adipose tissue (202.9 kDa [20]), erythrocytes (240 kDa [21]), placenta (240 kDa [22]) and granulocyte (263 kDa [50]) of human, liver (247.5 and 240 kDa [25, 51]) and milk (225 kDa [24]) of bovine, kidney (209 and 219 kDa [26, 52]) and erythrocytes (250 kDa [53]) of porcine, liver and erythrocytes of dog (230 kDa [32, 33]), goat liver (220 kDa [29]), horse liver (225 kDa [35]), sheep erythrocytes (242.1 kDa [36]) and rat liver (249 and 256 kDa [30, 31]). Also, a comparison of the molecular weight of CAT from microorganisms [12, 54], plants [16, 18] and insects [55, 56] showed that the molecular weight of camel liver CAT is relatively higher. On the other hand, the molecular weight of camel liver CAT was lower when compared to that of purified CAT from erythrocytes of human [23] and horse [57], goat lung [28] and microorganisms [11, 13, 58]. 2.2.2. Optimum temperature and pH The effect of temperature on the activity of CAT was investigated from 20 to 80°C (Figure 3A). It has been reported that the CAT partially purified from camel liver had a broad optimum temperature between 25 and 40°C [37]. In this study, the enzyme showed more than 80% of the activity in the temperature range from 20 to 50°C. The optimum temperature was found to be 45°C. The optimum temperature of CAT purified from mammalian species was reported to be 20°C in bovine milk [24], 30°C in sheep erythrocytes [36] and water buffalo liver [51] and 40°C in bovine liver [54, 59]. These reported optimum temperatures were lower than that of the present study. Also, the lower optimum temperature was found for purified CAT from toad liver [59], chicken erythrocytes [60], fish liver [61], insect [55], bacteria [62–64] and plants [16, 18, 52]. In contrast, the optimum temperature found in this study was lower when compared to that of bullfrog liver (50°C) [65], plant (50°C) [19], bacteria (60°C) [14] and fungi (70°C) [11]. We showed that the optimum temperature of purified CAT from camel liver was higher than that from mammalian species, our result could be explained by the fact that camel is able
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to live in the harsh desert conditions such as high temperature, direct exposure to sunlight and without drinking water for weeks. The effect of pH on the activity of CAT was analyzed under different buffer conditions (pH 6.0 to 9.0) (Figure 3B). The purified CAT was active over a broad pH range of 6.0 to 9.0 and retained more than 60% of its activity. The optimum pH for the enzyme was found to be 7.2, which was similar to that reported in literature. The optimum pH of purified CAT falls in the range from 5.0 to 9.0 in mammalian tissues [20, 24, 26, 29, 36, 37, 50, 51], from 4.0 to 12.0 in microorganisms [11–14, 66, 62], from 5.0 to 8.0 in plants [16, 18, 19, 52] and from 6.5 to 8.0 in insects [55, 56]. On the other hand, the CAT enzyme has been shown to exhibit sharp optimum pH [12–14, 16, 19, 24, 29, 36, 37, 51, 52, 55, 62] or a broad optimum pH [11, 18, 20, 26, 50, 56, 66]. 2.2.3. Effect of inhibitors, metal ions and reduced glutathione Some inhibitors were used to study their effect on the activity of CAT (Table 2). The enzyme was strongly inhibited by SDS and PMSF, and was fairly inhibited by β-ME, EDTA and DTT. From the effects of the thiol compounds, β-ME and DTT, the inhibition of CAT activity increased gradually. EDTA inhibited the CAT enzyme activity which indicates that it is metalloenzyme. These observations were similar to that obtained in other studies [16, 18, 28, 55, 60, 67]. The activity of CAT was also tested in the presence of various concentrations of ions (Figure 4). Increase in the CAT activity was observed with the increase in metal ion concentration of Ca2+, Co2+ and Ni2+, which they might serve as cofactors [16, 68], while Al3+, Cd2+ and Mg2+ inhibited the activity of CAT with the increase in the concentration of respective metal ions. These results agree with the published data [16, 69]. The oxidation of GSH by H2O2 has been reported [70, 71]. Figure 5 shows the effect of GSH concentration, from 0.5 to 5.0 mM, on non-enzymatic reaction and on CAT activity at constant H2O2 concentration (10 mM). The residual GSH was determined in the absence and presence of the CAT preparation. In the absence of CAT, increasing GSH concentration showed that the non-enzymatic reaction of the oxidation of GSH by H2O2 was higher. Although, relatively no effect was observed on the non-enzymatic reaction of the oxidation of GSH by H2O2 in the presence of CAT. On the other hand, the activity of CAT was apparently unchanged in the presence of GSH. Previous study observed the inactivation of CAT from mussels, marine plants and beef kidney in the presence of GSH [72]. GSH is a major antioxidant and required 9
as a cofactor for some enzymes involved in oxidative stress [73]. Our results indicated that camel liver CAT plays an important role in protecting GSH from H2O2 oxidation. Consequently, GSH might play an important role in protecting camel from free radical damage and in the adaptation to their life in the desert. 2.2.4. Kinetic parameters The kinetic parameters of the purified CAT from camel liver were determined with nonlinear regression using GraphPad Prism software version 7 (Figure 6). The calculated parameters are summarized in Table 3. The Km and Vmax values for H2O2 were calculated to be 37.31 mM and 6185157 U/mg, respectively. Km values of the purified CAT from mammalian species were found to be 38.9 mM in human white adipose tissue [20], 58 mM [51], 70 mM [74], 93 mM [10] and 33.3 × 102 M [75] in bovine liver, 80 mM in human erythrocytes [10], 70 and 110 mM in lung and liver of goat [28, 76], respectively. The lower Km value of camel liver CAT compared to that reported in mammalian species shows a greater affinity of camel liver CAT towards H2O2. Also, the lower Km value (22.7 mM H2O2/ml) was reported for the CAT partially purified from camel liver [37]. The higher affinity for H2O2 found in the present study, probably mean that under oxidative stress there is an acceleration in the response of the enzyme for H2O2 destruction, these may play an important role in the adaptation of camel to their arid life in the desert by protecting itself from potentially lethal effects of oxidative stress. The effect of varying concentration of NaN3 at three different concentrations of H2O2 was tested (Figure 6A). The inhibition type of camel liver CAT by NaN3 was found to be noncompetitive type with the Ki value of 14.43 µM. It is well known that NaN3 was found to be the most potent inhibitor of CAT enzyme. The kinetic parameters of the inhibitory effect of NaN3 on purified CAT from mammalian sources have not been explored earlier. Although there are a few reports which discuss about the inhibition effect of CAT by NaN3 from microorganisms [12, 13, 58, 66, 77], plants [17, 78] and insect [69]. As for Km value, the lower Ki value of camel liver CAT compared to that of fungi (6.1 mM) [13] and insect (0.28 mM) [69] shows a greater affinity of camel CAT towards NaN3. The effect of varying concentration of NaN3 and keeping H2O2 concentration constant at 10 mM was tested (Figure 6B). The IC50 was found to be 16.71 µM and the maximum inhibition of the enzyme was achieved by 400 µM NaN3. The IC50 of camel liver CAT was higher compared to that of bovine liver and human erythrocytes (1.5 µM) [10]. Although, compared 10
with that found in microorganisms (20 µM [12], 60 µM [66], 150 µM [77] and 330 µM [58]), plants (20 µM [17, 78]) and insects (400 µM [69]), the IC50 of camel liver CAT was lower. The Hill slope was found to be 1.34 indicated the existence of binding sites for NaN3 on the purified CAT from camel liver. 3. Conclusion This study presents, for the first time, the purification of CAT from camel liver by zinc chelate affinity chromatography using pH gradient elution. This purification procedure gives a better separation. A higher specific activity of purified enzyme was obtained. The comparative characterization of the purified enzyme has shown that camel liver CAT has some different properties than that of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of GSH from H2O2 oxidation and higher affinity for H2O2 and NaN3, these unique properties of camel liver CAT could be explained by the fact that camel is able to live in the specific ecosystem in the desert. On the other hand, we are reported that the camels were exposed to heavy metals (cadmium and lead) either through their feed (pasture) or drinking water [79], this could result in oxidative stress if the antioxidant system is unable to buffer the increase in the production of the free radicals. Our studies are important steps towards understanding the antioxidant defense system in camel, a unique creature among the other domestic species that is adapted for desert life. The camel has its place for the future, notably with the climatic changes. Future studies will be performed on the metabolism of camels by studying some of the enzymes involved in the metabolism of ROS and oxidative stress, to better understand the mechanisms involved in the resistance to stress induced by the life in the desert (dehydration, high temperatures, shortage and low nutritional level). Conflicts of interest The authors declare no conflicts of interest. Acknowledgments The authors thank Atatürk University (Turkey) and University Hassan First (Morocco) for their financial support. References
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17
Figure captions: Figure 1: (A) The chromatographic profile of CAT from camel liver using zinc chelate affinity chromatography. (B) CAT activity staining on native PAGE. (C) SDS-PAGE analysis of CAT from camel liver. Lane 1, molecular weight standard; lane 2, affinity chromatography; lane 3, ion exchange chromatography; lane 4, ethanol-chloroform treatment. (D) Sephacryl S-200 column chromatography of camel liver CAT. To estimate the molecular weight of the CAT peak, the same column was calibrated with molecular weight of standard proteins as described under Materials and methods. Figure 2: (A) Molecular weight determination of camel liver CAT by gel filtration on a Sephacryl S-200 column (1.75 × 37 cm). Ve is the elution volume of each protein and Vo is the void volume determined using blue dextran. (B) Determination of the subunit molecular weight of camel liver CAT by SDS-PAGE. Experimental conditions were used as described under Materials and methods. The red square indicates the CAT enzyme. Figure 3: Effect of temperature (A) and pH (B) on activity of CAT from camel liver. Figure 4: Effect of metal ions on activity of CAT from camel liver. Figure 5: Oxidation of GSH by H2O2 (H2O2 + GSH, gray line) and effect of GSH on CAT activity (H2O2 + GSH + CAT, pink line). The results of CAT activity were given as percentage of control (100%) without GSH (blue line). CAT activity and residual GSH were determined as described under Materials and methods. Figure 6: (A) Nonlinear regression plot to determine Km and Vmax of CAT for H2O2, and Ki and the type of inhibition of CAT for NaN3. (B) Determination of IC50 and Hill slope of CAT by varying concentrations of NaN3. All kinetic parameters were calculated using GraphPad Prism software version 7.
18
Figure 1.
19
Figure 2.
20
Figure 3.
21
Figure 4.
22
Figure 5.
23
Figure 6. Table 1. Summary of CAT purification from camel liver. Volume Step ml Ethanol-
Protein mg/ml
CAT activity U*/ml
U
Specific activity U/mg
Purification
Yield
-fold
%
20
Chloroform
549.81 3085458.72 61709174.31
5611.83
1.00 100.00
treatment Ion exchange
30
chromatography Affinity chromatography
17
7.51
146238.53
0.04
42337.92
4387155.96
19478.24
3.47
7.11
719744.65 1132539.37
201.81
1.17
* One unit of CAT activity was defined as the calculated consumption of 1 µmol of H2O2 per min at 25°C.
24
Table 2. Effect of inhibitors on activity of CAT from camel liver. Reagent
Control β-ME
EDTA
DTT
PMSF
SDS
Final concentration
Residual activity
(mM)
(%)
–
100.00
2.0
98.47
5.0
77.39
2.0
91.45
5.0
70.15
2.0
88.20
5.0
81.47
2.0
78.00
5.0
12.18
2.0
48.53
5.0
0.00
25
Table 3. Summary of kinetic parameters for camel liver CAT. H2O2
Km (mM) Vmax (U/mg)
NaN3
37.31 ± 7.89 6185157 ± 8.16
Ki (µM)
14.43 ± 1.05
IC50 (µM)
16.71 ± 0.92
Hill slope
1.34 ± 0.08
26
27
28
S1570-0232(17)31251-5 https://doi.org/10.1016/j.jchromb.2017.10.052 CHROMB 20882
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
19-7-2017 17-10-2017 26-10-2017
Please cite this article as: Abdelbasset Chafik, Abdelkhalid Essamadi, Safinur Yildirim C ¸ elik, Ahmet Mavi, Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties, Journal of Chromatography B https://doi.org/10.1016/j.jchromb.2017.10.052 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties Abdelbasset Chafika*, Abdelkhalid Essamadia, Safinur Yildirim Çelikb, Ahmet Mavic a
Laboratory of Biochemistry and Neuroscience, Team of Applied Biochemistry and
Toxicology, Faculty of Science and Technology, University Hassan First, 577, Settat, Morocco b
c
College of Education, Bayburt University, 69000, Bayburt, Turkey
Chemistry Education, Kazim Karabekir Education Faculty, Atatürk University, 25240, Erzurum, Turkey
*The author to whom correspondence should be addressed Abdelbasset Chafik Laboratory of Biochemistry and Neuroscience, Team of Applied Biochemistry and Toxicology, Faculty of Science and Technology, University Hassan First, 577, Settat, Morocco E-mail: [email protected] Phone: +212 762560876
Highlights
The camel is often subjected to intense environmental stress in the desert. Catalase plays a key role in protecting cells against oxidative stress. Catalase from camel liver was purified by zinc chelate affinity chromatography. The properties of purified catalase were different comparing to those of mammals. The catalase could play an important role in the camel's adaptation to its milieu.
Abstract: Climate change and increasing temperatures are global concerns. Camel (Camelus dromedarius) lives most of its life under high environmental stress in the desert and represent ideal model for studying desert adaptation among mammals. Catalase plays a key role in protecting cells against oxidative stress. For the first time, catalase from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution, a better separation was obtained. A purification fold of 201.81 with 1.17% yield and a high 1
specific activity of 1132539.37 U/mg were obtained. The native enzyme had a molecular weight of 268 kDa and was composed of four subunits of equal size (65 kDa). The enzyme showed optimal activity at a temperature of 45°C and pH 7.2. Thiol reagents, β-Mercaptoethanol and D,L-Dithiothreitol, inhibited the enzyme activity. The enzyme was inhibited by Al3+, Cd2+ and Mg2+, whereas Ca2+, Co2+ and Ni2+ stimulated the catalase activity. Reduced glutathione has no effect on catalase activity. The Km and Vmax of the enzyme for hydrogen peroxide were 37.31 mM and 6185157 U/mg, respectively. Sodium azide inhibited the enzyme noncompetitively with Ki value of 14.43 µM, the IC50 was found to be 16.71 µM. The properties of camel catalase were different comparing to those of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of reduced glutathione from hydrogen peroxide oxidation and higher affinity for hydrogen peroxide and sodium azide, these could be explained by the fact that camel is able to live in the intense environmental stress in the desert.
Abbreviations: ROS: Reactive oxygen species; CAT: Catalase; H2O2: Hydrogen peroxide; MCAC: Metal chelate affinity chromatography; GSH: Reduced glutathione; DTNB: 5,5′Dithiobis(2-nitrobenzoic acid); β-ME: β-Mercaptoethanol; DTT: D,L-Dithiothreitol; PMSF: Phenylmethylsulfonylfluoride; EDTA: Ethylenediaminetetraacetate; NaN3: Sodium azide.
Keywords: Camel; Camelus dromedarius; Desert; Catalase; Affinity chromatography; pH gradient elution.Introduction The one-humped camel (Camelus dromedarius), is among the mammals domesticated by humans for their needs in the desert, as it is a multipurpose animal used for production, leisure, transport, agricultural work or treatment of diseases [1, 2]. It is well known that camel has some biochemical, anatomical and physiological peculiarities due to their adaptation to desert life and differ it from other mammalian species [3, 4], although much less is known about physiological and biochemical studies of enzymes involved in the metabolism of reactive oxygen species (ROS) and oxidative stress in this animal, especially with the climatic changes and increasing temperatures [5]. The camel is continuously exposed to heat, solar radiation, and lack of water and food [6, 7]. This specific ecosystem could result in a disturbance in the balance between the production 2
of ROS and antioxidant defenses, and therefore causing damage of important biomolecules such as DNA, proteins and enzymes [8]. Generally, living organisms are equipped with both enzymatic and non-enzymatic antioxidant defense mechanisms to protect themselves against ROS [9]. Catalase (CAT, EC 1.11.1.6) is one of the principle antioxidant enzymes, which plays a key role in protecting cells from oxidative damage. It catalyzes the conversion of hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2) [10]. CAT has been purified and characterized from a large number of microorganisms [11–15] and plants [16–19]. In mammalian species, the enzyme has been purified and characterized from human [20–23], bovine [24, 25], porcine [26, 27], goat [28, 29], rat [30, 31], dog [32, 33], mouse [34], horse [35] and sheep [36]. However, information on CAT from camel is limited. Until now only one report has appeared on the partial purification and characterization of this enzyme from camel liver [37]. On the other hand, numerous purification procedures of CAT are frequently used ion exchange chromatography and gel filtration chromatography. Purification of CAT from mammalians [21, 25, 34], plants [16] and microorganisms [12, 13, 15] using metal chelate affinity chromatography (MCAC) is limited. Step-wise increase in the concentration of imidazole [12, 13, 15, 16, 25, 34] or linear gradient of histidine [21] were used to elute the adsorbed CAT from MCAC. The method of purification of CAT by MCAC using pH gradient elution has not been reported. This paper describes for the first time the purification of CAT from camel liver by MCAC using pH gradient elution, and compares some of its properties with those of other CATs. The study of this protein biomarker will contribute to understanding some of the mechanisms involved in the resistance to stress induced by the life in the desert. 1. Materials and methods 1.1. Chemicals DEAE-Sepharose, imminodiacetic acid immobilized Sepharose 6B, Sephacryl S-200, gel filtration markers kit for protein molecular weights 12–200 kDa, 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB), reduced glutathione (GSH), phenylmethylsulfonylfluoride (PMSF) and D,LDithiothreitol (DTT) were purchased from Sigma Aldrich. PageRuler™ Plus Prestained Protein Ladder 10–250 kDa were purchased from ThermoFisher Scientific. All other chemicals were of analytical grade. 1.2. Purification of CAT 3
1.2.1. Ethanol-Chloroform treatment Fresh camel liver was obtained from municipal slaughterhouse of Casablanca–Morocco. Approximately 73 g of liver was homogenized in 2 volumes of cold potassium phosphate buffer (20 mM, pH 7.0) using a Waring blender. The mixture was then centrifuged at 3500 × g for 20 min at 4°C. The hemoglobin was precipitated by chloroform-ethanol treatment according to Tsuchihashi [38]. With stirring, 0.25 volume of ethanol and 0.15 volume of chloroform, precooled in a freezer (-24°C), were added rapidly to the lysate. After 15 min, the mixture was centrifuged at 3500 × g for 60 min at 4°C. Thereafter, the supernatant was dialyzed against four changes of 20 mM potassium phosphate buffer pH 7.0, 20 volumes each, over a period of eight hours at 4°C. 1.2.2. DEAE-Sepharose ion exchange chromatography The precipitate which formed during dialysis was removed by centrifugation and the supernatant was loaded on a DEAE-Sepharose column (2.5 × 10 cm) pre-equilibrated with the same buffer as that used for dialysis. The adsorbed proteins were eluted at room temperature with a linear gradient of 0 to 0.5 M KCl prepared in the equilibration buffer at a flow rate of 60 ml/h. Fractions of 2 ml were collected and the fractions containing CAT activity were subjected to zinc chelate affinity chromatography. 1.2.3. Zinc chelate affinity chromatography An affinity column (1.6 × 9 cm) was packed with imminodiacetic acid immobilized Sepharose 6B and activated with 20 mM ZnSO4.7H2O solution. A second column (1.4 × 4 cm) was packed with the same gel, which did not receive the zinc solution, and connected to the bottom of the column containing the metal-activated gel. The second column served as a guard to adsorb leakage of zinc ions from the first column. The sample was applied to the first column equilibrated with 20 mM potassium phosphate buffer pH 7.0 containing 0.1 M NaCl. After washing with the same buffer, CAT was eluted at room temperature with a linear gradient of pH from 20 mM potassium phosphate buffer pH 7.0 to 20 mM citrate buffer pH 5.0, both buffers containing 0.1 M NaCl. A constant flow-rate of 60 ml/h was maintained throughout the procedure. The activity of CAT was determined in all the eluted fractions of 1 ml and activitycontaining fractions were collected and stored at -24°C until use. 1.2.4. Homogeneity of the purified enzyme 4
Homogeneity of the purified enzyme was confirmed by electrophoretic analysis using a vertical slab gel apparatus. Native gel electrophoresis was carried out with 7.5% PAGE according to Davis [39]. SDS-PAGE was performed with 12% polyacrylamide gel according to Laemmli [40]. The proteins were stained with coomassie brilliant blue G-250. 1.3. Characterization of CAT 1.3.1. Molecular weight determination The native molecular weight of the purified enzyme was estimated using Sephacryl S-200 (1.75 × 37 cm) gel filtration column. The column was equilibrated with 50 mM Tris-HCl (pH 7.5) containing 0.1 M KCl and calibrated with the following standard proteins: Cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), albumin (66 kDa), alcohol dehydrogenase (150 kDa) and β-Amylase (200 kDa). CAT was eluted with the above-mentioned buffer at a flow rate of 0.5 ml/min and the enzyme activity was determined in the collected fractions of 1 ml. The molecular weight of the subunits of the enzyme was estimated by SDS-PAGE according to the method of Weber and Osborn [41], using 12% polyacrylamide gel with a current of 50 mA for approximately 4 h. Molecular weight marker proteins used for SDS-PAGE were from commercial grade with molecular mass ranging from 10 to 250 kDa. Protein bands were visualized in gel by staining with 0.3% coomassie brilliant blue G-250. 1.3.2. Optimum temperature and pH The optimum temperature of the enzymatic reaction of purified enzyme was determined over a temperature range from 20 to 80°C. To determine optimum pH for enzymatic reaction, activity of purified enzyme was measured using two different buffer systems in two different pH ranges. A 0.05 M phosphate buffer was used for pH 6.0–8.0 intervals and a 0.05 M Tris-HCl buffer was used for pH 8.6–9.0 intervals. 1.3.3. Effect of inhibitors, metal ions and reduced glutathione The effect of inhibitors, metal ions and reduced glutathione (GSH) on the activity of purified enzyme were investigated. β-Mercaptoethanol (β-ME), ethylenediaminetetraacetic acid (EDTA), D,L-Dithiothreitol (DTT), phenylmethylsulfonylfluoride (PMSF) and sodium dodecyl sulfate (SDS) were tested at a final working concentration of 2 and 5 mM. The purified enzyme was incubated with a final working concentration of 1, 2 and 5 mM of salts of metal ions (chlorides of Al3+, Ca2+, Cd2+, Co2+, Mg2+ and Ni2+) to tested the effect of these metal ions 5
on the activity of purified enzyme. Since non-enzymatic reaction between GSH and H2O2 occurred [42, 43], the effect of GSH on the non-enzymatic reaction and on CAT activity was studied by varying the concentration of GSH from 0.5 to 5.0 mM. The residual GSH was determined according to the procedure of Sedlak and Lindsay [44] based on Ellman's reagent [45], with slight modifications. After 1 min, according to the assay system of CAT activity, 250 µl of 0.04% 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) prepared in 0.4 M phosphate buffer pH 7.0 was added immediately in the cuvette. The optical density measured at 412 nm after 2 min, is directly proportional to GSH concentration in the sample. The GSH concentration in sample was determined by reference to the standard curve of GSH. 1.3.4. Kinetic parameters Purified enzyme was used to calculate Km and Vmax of CAT for H2O2, and Ki, IC50 and Hill slope of CAT for sodium azide (NaN3). CAT activity was measured as described in assay system. The Km, Vmax and Ki were determined by varying concentrations of NaN3, from 0 to 80 µM, and keeping H2O2 concentration constant at 10, 20 and 30 mM; experiments to determine IC50 and Hill slope were carried out with varying concentrations of NaN3, from 0 to 500 µM, and keeping H2O2 concentration constant at 10 mM. Data were analyzed with nonlinear regression using GraphPad Prism software version 7. 1.3.5. Assay of CAT activity CAT activity was assayed according to the method of Aebi [46]. The assay was performed in a reaction mixture containing 10 mM H2O2 in 50 mM potassium phosphate buffer pH 7.0, and the reaction was started by addition of enzyme solution. CAT activity was determined spectrophotometrically by monitoring the decrease in H2O2 absorption at 240 nm for 1 min. One unit of CAT activity was defined as the calculated consumption of 1 µmol of H2O2 per min at 25°C. The molar extinction coefficient for H2O2 is 43.6 M-1 cm-1. The experiments were done in triplicate. Staining of CAT activity on native PAGE was performed according to the procedure of Woodbury et al. [47]. After electrophoresis, the gel was soaked for 10 min in 0.01% of H2O2. The gel was then stained with a reaction mixture containing 1% potassium ferricyanide and 1% ferric chloride. Achromatic bands of CAT appeared against a green background. 1.3.6. Protein determination 6
Protein concentrations were measured by the dye binding assay method of Bradford [48] using bovine serum albumin as a standard. 2. Results and discussion 2.1. Purification and homogeneity After precipitation of hemoglobin by ethanol-chloroform treatment, CAT from camel liver was purified by two steps of column chromatography using DEAE-Sepharose and zinc chelate affinity chromatography. CAT purified from mammalians [25, 34], plants [16] and microorganisms [12, 13, 15] using MCAC was generally eluted by step-wise using a specific eluent such as imidazole. For the first time, CAT from camel liver was purified by zinc chelate affinity chromatography using pH gradient elution. The CAT was eluted with a linear gradient of pH using 20 mM potassium phosphate buffer pH 7.0 and 20 mM citrate buffer pH 5.0, both buffers contain 0.1 M NaCl. As shown in Figure 1A, CAT was eluted between pH 6.13 and 5.28. A better separation was obtained using pH gradient elution, which removed a small amount of the contaminating proteins. In the same manner, contaminating proteins were separated using linear gradient of histidine [21]. The purification profile of CAT from camel liver is given in Table 1. The specific activity of the final preparation was 1132539.37 U/mg protein, which represents 201.81-fold purification with a 1.17% yield. The specific activity of the purified camel liver CAT was higher than that of purified CAT from different organs of human [20–23, 49], liver and milk of bovine [24, 25], porcine kidney [26], goat lung [28], liver and erythrocytes of dog [32, 33], mouse liver [34], sheep erythrocytes [36] and murine erythrocytes [49]. The purification fold of the purified CAT by MCAC was reported to be 4.2-fold in bovine liver [25], 12 and 44-fold in mouse liver [34], 38.7 and 161.8-fold in bacteria [12, 15], 23.3 and 106.8-fold in plant [16]. The purification fold achieved in this study (201.81-fold) was higher than that in reported studies. The 565-fold purification was reported in human erythrocytes CAT purified by MCAC [21]. CAT from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution. The final enzyme preparation was subjected to PAGE and SDSPAGE. The purified enzyme gave a single protein band when the gel was stained for CAT activity (Figure 1B) and for protein with coomassie brilliant blue G-250 (Figure 1C). Also, the purified enzyme was eluted from Sephacryl S-200 column as a single peak of enzyme activity (Figure 1D). 7
2.2. Characterization and some properties of purified CAT 2.2.1. Molecular weight estimation The native molecular weight of CAT eluted from Sephacryl S-200 column was found to be 267827 Da (Figure 2A). The subunit molecular weight of the enzyme was estimated to be 64597 Da from SDS-PAGE, as shown in Figure 2B. These results suggested that the camel liver CAT was tetramer composed of four identical subunits. The molecular weight of camel liver CAT was relatively higher than that of purified CAT, containing four identical subunits, from white adipose tissue (202.9 kDa [20]), erythrocytes (240 kDa [21]), placenta (240 kDa [22]) and granulocyte (263 kDa [50]) of human, liver (247.5 and 240 kDa [25, 51]) and milk (225 kDa [24]) of bovine, kidney (209 and 219 kDa [26, 52]) and erythrocytes (250 kDa [53]) of porcine, liver and erythrocytes of dog (230 kDa [32, 33]), goat liver (220 kDa [29]), horse liver (225 kDa [35]), sheep erythrocytes (242.1 kDa [36]) and rat liver (249 and 256 kDa [30, 31]). Also, a comparison of the molecular weight of CAT from microorganisms [12, 54], plants [16, 18] and insects [55, 56] showed that the molecular weight of camel liver CAT is relatively higher. On the other hand, the molecular weight of camel liver CAT was lower when compared to that of purified CAT from erythrocytes of human [23] and horse [57], goat lung [28] and microorganisms [11, 13, 58]. 2.2.2. Optimum temperature and pH The effect of temperature on the activity of CAT was investigated from 20 to 80°C (Figure 3A). It has been reported that the CAT partially purified from camel liver had a broad optimum temperature between 25 and 40°C [37]. In this study, the enzyme showed more than 80% of the activity in the temperature range from 20 to 50°C. The optimum temperature was found to be 45°C. The optimum temperature of CAT purified from mammalian species was reported to be 20°C in bovine milk [24], 30°C in sheep erythrocytes [36] and water buffalo liver [51] and 40°C in bovine liver [54, 59]. These reported optimum temperatures were lower than that of the present study. Also, the lower optimum temperature was found for purified CAT from toad liver [59], chicken erythrocytes [60], fish liver [61], insect [55], bacteria [62–64] and plants [16, 18, 52]. In contrast, the optimum temperature found in this study was lower when compared to that of bullfrog liver (50°C) [65], plant (50°C) [19], bacteria (60°C) [14] and fungi (70°C) [11]. We showed that the optimum temperature of purified CAT from camel liver was higher than that from mammalian species, our result could be explained by the fact that camel is able
8
to live in the harsh desert conditions such as high temperature, direct exposure to sunlight and without drinking water for weeks. The effect of pH on the activity of CAT was analyzed under different buffer conditions (pH 6.0 to 9.0) (Figure 3B). The purified CAT was active over a broad pH range of 6.0 to 9.0 and retained more than 60% of its activity. The optimum pH for the enzyme was found to be 7.2, which was similar to that reported in literature. The optimum pH of purified CAT falls in the range from 5.0 to 9.0 in mammalian tissues [20, 24, 26, 29, 36, 37, 50, 51], from 4.0 to 12.0 in microorganisms [11–14, 66, 62], from 5.0 to 8.0 in plants [16, 18, 19, 52] and from 6.5 to 8.0 in insects [55, 56]. On the other hand, the CAT enzyme has been shown to exhibit sharp optimum pH [12–14, 16, 19, 24, 29, 36, 37, 51, 52, 55, 62] or a broad optimum pH [11, 18, 20, 26, 50, 56, 66]. 2.2.3. Effect of inhibitors, metal ions and reduced glutathione Some inhibitors were used to study their effect on the activity of CAT (Table 2). The enzyme was strongly inhibited by SDS and PMSF, and was fairly inhibited by β-ME, EDTA and DTT. From the effects of the thiol compounds, β-ME and DTT, the inhibition of CAT activity increased gradually. EDTA inhibited the CAT enzyme activity which indicates that it is metalloenzyme. These observations were similar to that obtained in other studies [16, 18, 28, 55, 60, 67]. The activity of CAT was also tested in the presence of various concentrations of ions (Figure 4). Increase in the CAT activity was observed with the increase in metal ion concentration of Ca2+, Co2+ and Ni2+, which they might serve as cofactors [16, 68], while Al3+, Cd2+ and Mg2+ inhibited the activity of CAT with the increase in the concentration of respective metal ions. These results agree with the published data [16, 69]. The oxidation of GSH by H2O2 has been reported [70, 71]. Figure 5 shows the effect of GSH concentration, from 0.5 to 5.0 mM, on non-enzymatic reaction and on CAT activity at constant H2O2 concentration (10 mM). The residual GSH was determined in the absence and presence of the CAT preparation. In the absence of CAT, increasing GSH concentration showed that the non-enzymatic reaction of the oxidation of GSH by H2O2 was higher. Although, relatively no effect was observed on the non-enzymatic reaction of the oxidation of GSH by H2O2 in the presence of CAT. On the other hand, the activity of CAT was apparently unchanged in the presence of GSH. Previous study observed the inactivation of CAT from mussels, marine plants and beef kidney in the presence of GSH [72]. GSH is a major antioxidant and required 9
as a cofactor for some enzymes involved in oxidative stress [73]. Our results indicated that camel liver CAT plays an important role in protecting GSH from H2O2 oxidation. Consequently, GSH might play an important role in protecting camel from free radical damage and in the adaptation to their life in the desert. 2.2.4. Kinetic parameters The kinetic parameters of the purified CAT from camel liver were determined with nonlinear regression using GraphPad Prism software version 7 (Figure 6). The calculated parameters are summarized in Table 3. The Km and Vmax values for H2O2 were calculated to be 37.31 mM and 6185157 U/mg, respectively. Km values of the purified CAT from mammalian species were found to be 38.9 mM in human white adipose tissue [20], 58 mM [51], 70 mM [74], 93 mM [10] and 33.3 × 102 M [75] in bovine liver, 80 mM in human erythrocytes [10], 70 and 110 mM in lung and liver of goat [28, 76], respectively. The lower Km value of camel liver CAT compared to that reported in mammalian species shows a greater affinity of camel liver CAT towards H2O2. Also, the lower Km value (22.7 mM H2O2/ml) was reported for the CAT partially purified from camel liver [37]. The higher affinity for H2O2 found in the present study, probably mean that under oxidative stress there is an acceleration in the response of the enzyme for H2O2 destruction, these may play an important role in the adaptation of camel to their arid life in the desert by protecting itself from potentially lethal effects of oxidative stress. The effect of varying concentration of NaN3 at three different concentrations of H2O2 was tested (Figure 6A). The inhibition type of camel liver CAT by NaN3 was found to be noncompetitive type with the Ki value of 14.43 µM. It is well known that NaN3 was found to be the most potent inhibitor of CAT enzyme. The kinetic parameters of the inhibitory effect of NaN3 on purified CAT from mammalian sources have not been explored earlier. Although there are a few reports which discuss about the inhibition effect of CAT by NaN3 from microorganisms [12, 13, 58, 66, 77], plants [17, 78] and insect [69]. As for Km value, the lower Ki value of camel liver CAT compared to that of fungi (6.1 mM) [13] and insect (0.28 mM) [69] shows a greater affinity of camel CAT towards NaN3. The effect of varying concentration of NaN3 and keeping H2O2 concentration constant at 10 mM was tested (Figure 6B). The IC50 was found to be 16.71 µM and the maximum inhibition of the enzyme was achieved by 400 µM NaN3. The IC50 of camel liver CAT was higher compared to that of bovine liver and human erythrocytes (1.5 µM) [10]. Although, compared 10
with that found in microorganisms (20 µM [12], 60 µM [66], 150 µM [77] and 330 µM [58]), plants (20 µM [17, 78]) and insects (400 µM [69]), the IC50 of camel liver CAT was lower. The Hill slope was found to be 1.34 indicated the existence of binding sites for NaN3 on the purified CAT from camel liver. 3. Conclusion This study presents, for the first time, the purification of CAT from camel liver by zinc chelate affinity chromatography using pH gradient elution. This purification procedure gives a better separation. A higher specific activity of purified enzyme was obtained. The comparative characterization of the purified enzyme has shown that camel liver CAT has some different properties than that of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of GSH from H2O2 oxidation and higher affinity for H2O2 and NaN3, these unique properties of camel liver CAT could be explained by the fact that camel is able to live in the specific ecosystem in the desert. On the other hand, we are reported that the camels were exposed to heavy metals (cadmium and lead) either through their feed (pasture) or drinking water [79], this could result in oxidative stress if the antioxidant system is unable to buffer the increase in the production of the free radicals. Our studies are important steps towards understanding the antioxidant defense system in camel, a unique creature among the other domestic species that is adapted for desert life. The camel has its place for the future, notably with the climatic changes. Future studies will be performed on the metabolism of camels by studying some of the enzymes involved in the metabolism of ROS and oxidative stress, to better understand the mechanisms involved in the resistance to stress induced by the life in the desert (dehydration, high temperatures, shortage and low nutritional level). Conflicts of interest The authors declare no conflicts of interest. Acknowledgments The authors thank Atatürk University (Turkey) and University Hassan First (Morocco) for their financial support. References
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Figure captions: Figure 1: (A) The chromatographic profile of CAT from camel liver using zinc chelate affinity chromatography. (B) CAT activity staining on native PAGE. (C) SDS-PAGE analysis of CAT from camel liver. Lane 1, molecular weight standard; lane 2, affinity chromatography; lane 3, ion exchange chromatography; lane 4, ethanol-chloroform treatment. (D) Sephacryl S-200 column chromatography of camel liver CAT. To estimate the molecular weight of the CAT peak, the same column was calibrated with molecular weight of standard proteins as described under Materials and methods. Figure 2: (A) Molecular weight determination of camel liver CAT by gel filtration on a Sephacryl S-200 column (1.75 × 37 cm). Ve is the elution volume of each protein and Vo is the void volume determined using blue dextran. (B) Determination of the subunit molecular weight of camel liver CAT by SDS-PAGE. Experimental conditions were used as described under Materials and methods. The red square indicates the CAT enzyme. Figure 3: Effect of temperature (A) and pH (B) on activity of CAT from camel liver. Figure 4: Effect of metal ions on activity of CAT from camel liver. Figure 5: Oxidation of GSH by H2O2 (H2O2 + GSH, gray line) and effect of GSH on CAT activity (H2O2 + GSH + CAT, pink line). The results of CAT activity were given as percentage of control (100%) without GSH (blue line). CAT activity and residual GSH were determined as described under Materials and methods. Figure 6: (A) Nonlinear regression plot to determine Km and Vmax of CAT for H2O2, and Ki and the type of inhibition of CAT for NaN3. (B) Determination of IC50 and Hill slope of CAT by varying concentrations of NaN3. All kinetic parameters were calculated using GraphPad Prism software version 7.
18
Figure 1.
19
Figure 2.
20
Figure 3.
21
Figure 4.
22
Figure 5.
23
Figure 6. Table 1. Summary of CAT purification from camel liver. Volume Step ml Ethanol-
Protein mg/ml
CAT activity U*/ml
U
Specific activity U/mg
Purification
Yield
-fold
%
20
Chloroform
549.81 3085458.72 61709174.31
5611.83
1.00 100.00
treatment Ion exchange
30
chromatography Affinity chromatography
17
7.51
146238.53
0.04
42337.92
4387155.96
19478.24
3.47
7.11
719744.65 1132539.37
201.81
1.17
* One unit of CAT activity was defined as the calculated consumption of 1 µmol of H2O2 per min at 25°C.
24
Table 2. Effect of inhibitors on activity of CAT from camel liver. Reagent
Control β-ME
EDTA
DTT
PMSF
SDS
Final concentration
Residual activity
(mM)
(%)
–
100.00
2.0
98.47
5.0
77.39
2.0
91.45
5.0
70.15
2.0
88.20
5.0
81.47
2.0
78.00
5.0
12.18
2.0
48.53
5.0
0.00
25
Table 3. Summary of kinetic parameters for camel liver CAT. H2O2
Km (mM) Vmax (U/mg)
NaN3
37.31 ± 7.89 6185157 ± 8.16
Ki (µM)
14.43 ± 1.05
IC50 (µM)
16.71 ± 0.92
Hill slope
1.34 ± 0.08
26
27
28