الفهرس 
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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