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Abstract This study aims at production of superoxide dismutase from the kidney of the common, locally available mammals as a rich source. Therefore, three domestic mammals were selected; water buffalo Bubalus bubalis, camel Camelus dromedaries and sheep Ovis aries. The level of superoxide dismutase activity was detected in the n-butanol extract of their kidneys and expressed as specific activity (unit / mg protein). Both the buffalo kidney cortex (297 ± 0.495 units / mg protein) and sheep kidney cortex (238 ± 0.604 units / mg protein) showed low and almost similar specific activity of the superoxide dismutase. In comparison, the camel kidney cortex contained the highest level of the superoxide dismutase specific activity (371± 0.2944 units / mg protein). The chromatographic patterns of the kidney superoxide dismutase of buffalo, camel and sheep were compared by using their elution profiles from the DEAE-cellulose column. All of the elution profiles revealed the presence of two major peaks of superoxide dismutase activity. The isoenzymes are designated BKSOD1 and BKSOD2 for buffalo, CKSOD1 and CKSOD2 for camel and SKSOD1 and SKSOD2 for sheep. The superoxide dismutase isoenzyme pattern of the three mammalian kidneys indicated the presence of three isoenzymes in camel kidney and two isoenzymes in both buffalo and sheep kidneys by native PAGE. Three different protein patterns were monitored by the native PAGE from the three mammals’ kidney. A simple and reproducible purification procedure is given involved n-butanol extraction, acetone precipitation, anion exchange chromatography on DEAE-cellulose column and gel filtration through Sephacryl S-300 column. 1- Water buffalo kidney superoxide dismutase isoenzymes The starting specific activity in the n-butanol extract was found to be 264.36 unit / mg protein. Most of the superoxide dismutase activity was precipitated with 1 volume of prechilled acetone. More than 83 % of the superoxide dismutase activity was recovered in the acetone fraction and the specific activity of the enzyme was increased more than 1.41-fold. Two major peaks exhibited the superoxide dismutase activity were resolved on DEAE-cellulose column and eluted with 0.0 M and 0.1 M NaCl and designated BKSOD1 and BKSOD2 respectively. The buffalo kidney superoxide dismutase specific activity of the pooled fractions of the two peaks BKSOD1 and BKSOD2 were increased 2.82 and 2.65 fold over the n-butanol extract with recovery of 42.98 % and 23.69 respectively. After the chromatography on the Sephacryl S-300 column, the specific activity of BKSOD1 was increased to 5113.29 units / mg protein which represent 19.34-fold purification over the n-butanol extract with 31.9 % yield. Also, the specific activity of BKSOD2 was increased to 4257.25 units / mg protein which represent 16.104-fold purification over the n-butanol extract with 17.96 % yield. By gel filtration, the molecular weight of the native form of BKSOD1 and BKSOD2 were calculated to be 63 ± 2.4 kDa and 66 ± 2.6 kDa respectively. Both BKSOD1 and BKSOD2 turned out to be homogeneous as judged by a single protein band on 7% native PAGE indicating the purity of these isoenzymes. Also the protein band of both BKSOD1 and BKSOD2 coincided with their enzyme activity band confirming that the single protein band is the enzyme band. By SDS-PAGE, the subunits molecular weight of BKSOD1 and BKSOD2 was estimated to be 63 ± 2.3 kDa and 66 ± 2.7 kDa respectively. These results indicate that both BKSOD1 and BKSOD2 are monomeric proteins composed of one subunit. By isoelectrofocusing, both BKSOD1 and BKSOD2 showed a single molecular species with an isoelectric point (pI) value of 6.7- 6.9 and 5.9- 6.1 respectively. Both BKSOD1 and BKSOD2 displayed an optimum activity at pH 7.6. The activity of the BKSOD1 is increased about 1.1, 1.19 and 1.45 fold in the presence of 2 mM NiCl2, ZnCl2 and CuCl2 respectively, and increased about 1.2, 1.73 and 2.76 fold in the presence of 5 mM NiCl2, ZnCl2 and CuCl2respectively. In contrast, CaCl2 and MgCl2 were found to be moderate inhibitors of BKSOD1, while FeCl2 is a potent inhibitor of BKSOD1. The activity of the BKSOD2 is increased 1.12, 1.6 and 2.18 fold in the presence of 2 mM MgCl2, CoCl2, and MnCl2 respectively, and increased 1.3, 1.74 and 2.6 fold in the presence of 5 mM MgCl2, CoCl2 and MnCl2. respectively. In contrast, FeCl2, ZnCl2 and CaCl2 are moderate inhibitors of BKSOD2 activity. Potassium cyanide and Hydrogen peroxide are found to be the most potent inhibitors of the activity of BKSOD1. Sodium azide is found to be a potent inhibitor of BKSOD2. Both isoenzymes were inhibited with EDTA, DL-dithiothreitol, â- Mercaptoethanol and 1, 10 phenanthroline. PMSF inhibited BKSOD2 Potassium dichromate is found to be a potent inhibitor of both isoenzymes. 2- Camel kidney superoxide dismutase isoenzymes The starting specific activity in the n-butanol extract was found to be 343.33 unit / mg protein. Most of the superoxide dismutase activity was precipitated with 1 volume of prechilled acetone. More than 88 % of the superoxide dismutase activity was recovered in the acetone fraction and the specific activity of the enzyme was increased more than 2.25-fold. Two major peaks exhibited the superoxide dismutase activity were resolved on DEAE-cellulose column and eluted with 0.0 M and 0.2 M NaCl and designated CKSOD1 and CKSOD2. The camel kidney superoxide dismutase specific activity of the pooled fractions of the two peaks CKSOD1 and CKSOD2 were increased 2.72 and 2.52 fold over the nbutanol extract with recovery of 48.72 % and 26.46 % respectively. The elution profile of CKSOD1 revealed the presence of two peaks of the enzyme activity eluted from the Sephacryl S-300 column (CKSOD1a and CKSOD1b), while the elution profile of CKSOD2 revealed the presence of one peak of the enzyme activity. After the chromatography on the sephacryl S-300 column, the specific activity of CKSOD1a was increased to 6379.31 units / mg protein which represents 18.58-fold purification over the n-butanol extract with 33.26 % yield and the specific activity of CKSOD1b was increased to 2450 units / mg protein which represent 7.136- fold purification over the n-butanol extract with 8.81 % yield. Also, the specific activity of CKSOD2 was increased to 5591units / mg protein which represent 16.28-fold purification over the n-butanol extract with 22.11 % yield. By gel filtration, the molecular weight of the native form of the three isoenzymes CKSOD1a, CKSOD1b and CKSOD2 were calculated to be 120 ± 1.3 kDa, 16 ± 1.8 kDa and 65 ± 1.3 kDa respectively. All isoenzymes CKSOD1a, CKSOD1b and CKSOD2 turned out to be homogeneous as judged by a single protein band on 7% native PAGE indicating the purity of these isoenzymes. Also the protein band of CKSOD1a, CKSOD1b and CKSOD2 coincided with their enzyme activity band confirming that the single protein band is the enzyme band. By SDSPAGE, the subunits molecular weight of CKSOD1a, CKSOD1b and CKSOD2 was estimated to be 60 ± 2.9 kDa, 16 ± 1.6 kDa and 65 ± 2.4 respectively. These results indicate that CKSOD1b and CKSOD2 are monomeric proteins composed of one subunit, while CKSOD1a is homodiameric protein composed of two identical subunits. By isoelectrofocusing, CKSOD1a, CKSOD1b and CKSOD2 showed a single molecular species with an isoelectric point (pI) value of 8-8.2 for CKSOD1a, 7.5-7.7 for CKSOD1b and 6.6-6.8 for CKSOD2. CKSOD1a, CKSOD1b and CKSOD2 displayed their optimum activity at pH 7.6. The activity of the CKSOD1a isoenzyme was increased about 1.11 fold in the presence of 2 mM MnCl2, and increased about 1.41 fold in the presence of 5 mM MnCl2. In contrast, FeCl2 was found to be potent inhibitor of CKSOD1a activity, while NiCl2 was a moderate inhibitor of CKSOD1a activity. The activity of the CKSOD1b isoenzyme was increased 1.11, 1.23, 1.34 and 1.6 fold in the presence of 2 mM CoCl2, CaCl2, ZnCl2 and CuCl2 respectively and was increased 1.11, 2.11 and 2.43 fold in the presence of 5 mM CaCl2, CuCl2 and ZnCl2 respectively. In contrast, NiCl2 was found to be a potent inhibitor of CKSOD1b activity. The activity of the CKSOD2 isoenzyme was increased 1.11, 1.12, 1.57 and 1.84 fold in the presence of 2 mM CoCl2, CaCl2, ZnCl2 and CuCl2 respectively and increased 1.16, 1.84 and 1.92 fold in the presence of 5 mM CoCl2, ZnCl2 and CuCl2. In contrast, NiCl2 and FeCl2 were found to be moderate inhibitors of CKSOD2 activity. Potassium cyanide and Hydrogen peroxide are found to be the most potent inhibitors of the CKSOD1b and CKSOD2. Sodium dodecyl sulphate is found to be a potent inhibitor of the activity of CKSOD1a. EDTA, DLdithiothreitol, â-Mercaptoethanol and 1, 10 phenanthroline inhibited the CKSOD1a, CKSOD1b and CKSOD2. PMSF inhibited CKSOD2 and iodoacetamide inhibited CKSOD1b. Potassium dichromate is found to be a potent inhibitor of CKSOD1a, CKSOD1b and CKSOD2. 3- Sheep kidney superoxide dismutase isoenzymes The starting specific activity in the n-butanol extract was found to be 205 unit / mg protein. Most of the superoxide dismutase activity was precipitated with 1 volume of prechilled acetone. More than 83 % of the superoxide dismutase activity was recovered in the acetone fraction and the specific activity of the enzyme was increased more than 1.69-fold. Two major peaks exhibited the superoxide dismutase activity were resolved on DEAE-cellulose column and eluted with 0.0 M and 0.1 M NaCl and designated SKSOD1 and SKSOD2. The Sheep kidney superoxide dismutase specific activity of the pooled fractions of the two peaks SKSOD1 and SKSOD2 were increased 2.7 and 2.41 fold over the n-butanol extract with recovery of 41.33 % and 18.52 % respectively. After the chromatography on the Sephacryl S-300 column, the specific activity of SKSOD1 was increased to 3539.77 units / mg protein which represent 17.27-fold purification over the n-butanol extract with 28.14 % yield. Also, the specific activity of SKSOD2 was increased to 2372.88 units / mg protein which represent 11.57-fold purification over the n-butanol extract with 12.65 % yield. By gel filtration, the molecular weight of the native form of SKSOD1 and SKSOD2 were calculated to be 63 ± 2.7 kDa and 120 ± 2.9 kDa respectively. Both SKSOD1 and SKSOD2 turned out to be homogeneous preparation as judged by a single protein band on 7 % native PAGE indicating the purity of these isoenzymes. Also the protein band of both SKSOD1 and SKSOD2 coincided with their enzyme activity band confirming that the single protein band is the enzyme band. By SDSPAGE, the subunits molecular weight of SKSOD1 and SKSOD2 was estimated to be 63 ± 2.3 kDa and 60 ± 2.8 kDa respectively. These results indicate that SKSOD1 is monomeric protein composed of one subunit, while SKSOD2 is homodiameric protein composed of two identical subunits. By isoelectrofocusing, both SKSOD1 and SKSOD2 showed a single molecular species with an isoelectric point (pI) value of 6.5-6.6 for SKSOD1 and 5.9-6.1 for SKSOD2. Both SKSOD1 and SKSOD2 displayed an optimum activity at pH 7.4 and 7.6 respectively. The activity of the SKSOD1 is increased about 1.1, 1.12 and 1.24 fold in the presence of 2 mM MgCl2, CaCl2, and MnCl2 respectively, and increased about 1.2, 1.31 and 1.42 fold in the presence of 5 mM MgCl2, CaCl2, and MnCl2 respectively. In contrast, NiCl2, and FeCl2 were found to be moderate inhibitors of SKSOD1 activity while CoCl2, ZnCl2 were exhibited nonsignificant effect on SKSOD1 activity. The activity of the SKSOD2 isoenzyme was increased about 1.14, 1.15, 1.28 and 1.47 folds in the presence of 2 mM CoCl2, MgCl2, ZnCl2 and CuCl2 respectively while increased about 1.21, 1.31, 1.54 and 1.64 folds in the presence of 5 mM of MgCl2, CoCl2, ZnCl2 and CuCl2 respectively. In contrast MnCl2 and NiCl2 were found to be moderate inhibitors of SKSOD2 activity, while FeCl2 exhibited non-significant effect. Potassium cyanide and Hydrogen peroxide are found to be the most potent inhibitors of the activity of SKSOD2. Sodium dodecyl sulphate, Sodium azide and DL-dithiothreitol are found to be potent inhibitors of the activity of SKSOD1. EDTA, â-Mercaptoethanol, 1, 10 phenanthroline and PMSF inhibited both SKSOD1 and SKSOD2. Both isoenzymes are resistant to Iodoacetamide. Potassium dichromate is found to be a potent inhibitor of both SKSOD1 and SKSOD2 isoenzymes |