الفهرس 
Diagnostic importance of superoxide dismutaseOnly 14 pages are availabe for public view
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Abstract
This study aims at production of superoxide dismutase
from the liver 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 livers and
expressed as specific activity (unit / mg protein). Both the
buffalo liver (128.203 ± 0.495 units / mg protein) and sheep
liver (116.963 ± 0.6039 units / mg protein) showed low and
almost similar specific activity of the superoxide dismutase. In
comparison, the camel liver contained the highest level of the
superoxide dismutase specific activity (214.742 ± 0.2944 units
/ mg protein). The chromatographic patterns of the liver
superoxide dismutase of buffalo, camel and sheep were
compared by using their elution profiles from the DEAEcellulose
column. All of the elution profiles revealed the
presence of two major peaks of superoxide dismutase activity.
The isoenzymes are designated BLSOD1 and BLSOD2 for
buffalo, CLSOD1 and CLSOD2 for camel and SLSOD1 and
SLSOD2 for sheep.
The superoxide dismutase isoenzyme pattern of the three
mammalian livers indicated the presence of three isoenzymes
in camel liver and two isoenzymes in both buffalo and sheep
- 222 - Summary
livers by native PAGE. Three different protein patterns were
monitored by the native PAGE from the three mammals’ liver.
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 liver superoxide dismutase isoenzymes
The starting specific activity in the n-butanol extract was
found to be 128.203 unit / mg protein. Most of the superoxide
dismutase activity was precipitated with 1 volume of
prechilled acetone. More than 79.4 % of the superoxide
dismutase activity was recovered in the acetone fraction and
the specific activity of the enzyme was increased more than
1.5-fold. Two major peaks exhibited the superoxide dismutase
activity were resolved on DEAE-cellulose column and eluted
with 0.05 M and 0.2 M NaCl and designated BLSOD1 and
BLSOD2 respectively. The buffalo liver superoxide dismutase
specific activity of the pooled fractions of the two peaks
BLSOD1 and BLSOD2 were increased 4.27 and 2.42 fold over
the n-butanol extract with recovery of 31.1 % and 13.8 %
respectively. After the chromatography on the sephacryl S-300
column, the specific activity of BLSOD1 was increased to
2673.9 units / mg protein which represent 20.79-fold
- 223 - Summary
purification over the n-butanol extract with 21.9 % yield. Also,
the specific activity of BLSOD2 was increased to 2562.30
units / mg protein which represent 19.92-fold purification over
the n-butanol extract with 11.2 % yield. By gel filtration, the
molecular weight of the native form of BLSOD1 and BLSOD2
were calculated to be 60 ± 2.4 kDa and 65 ± 2.6 kDa
respectively. Both BLSOD1 and BLSOD2 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 BLSOD1 and BLSOD2 coincided with
their enzyme activity band confirming that the single protein
band is the enzyme band. By SDS-PAGE, the subunits
molecular weight of BLSOD1 and BLSOD2 was estimated to
be 60 ± 2.3 kDa and 66 ± 2.7 kDa respectively. These results
indicate that both BLSOD1 and BLSOD2 are monomeric
proteins composed of one subunit. By isoelectrofocusing, both
BLSOD1 and BLSOD2 showed a single molecular species
with an isoelectric point (pI) value of 6.9-7.1 and 5.8-6.0
respectively. Both BLSOD1 and BLSOD2 displayed an
optimum activity at pH 7.6. The activity of the BLSOD1 is
increased about 1.04, 1.12, 1.17 and 1.28 fold in the presence
of 2 mM ZnCl2, CoCl2, NiCl2 and CuCl2 respectively, and
increased about 1.23, 1.36, 1.57 and 1.89 fold in the presence
of 5 mM NiCl2, CoCl2, ZnCl2 and CuCl2 respectively. In
- 224 - Summary
contrast, CaCl2 and MgCl2 were found to be moderate
inhibitors of BLSOD1, while FeCl2 is a potent inhibitor of
BLSOD1. The activity of the BLSOD2 is increased 1.49 fold
in the presence of 5 mM MnCl2. In contrast, CuCl2, CoCl2 and
FeCl2 are moderate inhibitors of BLSOD2. The activity of
both BLSOD1 and BLSOD2 are visualized on 7 % native
PAGE in the presence of 5 mM of CuCl2, ZnCl2 and MnCl2.
The presence of CuCl2 and ZnCl2 increased the BLSOD1 band
and did not affect the BLSOD2 while MnCl2 obviously
increased the BLSOD2 band. Potassium cyanide is found to be
the most potent inhibitor of the activity of BLSOD1. Sodium
dodecyl sulphate is found to be a potent inhibitor of BLSOD2.
Both isoenzymes were inhibited with EDTA, DL-dithiothreitol,
-Mercaptoethanol and 1, 10 phenanthroline. PMSF inhibited
BLSOD1 and iodoacetamide inhibited BLSOD2. Potassium
dichromate is found to be a potent inhibitor of both
isoenzymes. On 7 % native PAGE; BLSOD1 activity band
disappeared in the presence of 5 mM KCN. In the presence of
5 mM H2O2, both BLSOD1 and BLSOD2 activity bands
exhibited lower intensity than the control bands. 5 mM NaN3
lowered the intensity of the BLSOD1 activity band slightly
and the intensity of the BLSOD2 activity band sharply. The
presence of 5 mM SDS decreased the intensity of the BLSOD1
- 225 - Summary
activity band obviously and the intensity of the BLSOD2
activity band completely.
2- Camel liver superoxide dismutase isoenzymes
The starting specific activity in the n-butanol extract was
found to be 214.742 unit / mg protein. Most of the superoxide
dismutase activity was precipitated with 1 volume of
prechilled acetone. More than 85.7 % of the superoxide
dismutase activity was recovered in the acetone fraction and
the specific activity of the enzyme was increased more than
1.8-fold. Two major peaks exhibited the superoxide dismutase
activity were resolved on DEAE-cellulose column and eluted
with 0 M and 0.2 M NaCl and designated CLSOD1 and
CLSOD2. The camel liver superoxide dismutase specific
activity of the pooled fractions of the two peaks CLSOD1 and
CLSOD2 were increased 3.40 and 2.01 fold over the n-butanol
extract with recovery of 43.0 % and 19.5 % respectively. The
elution profile of CLSOD1 revealed the presence of two peaks
of the enzyme activity eluted from the sephacryl S-300 column
(CLSOD1a and CLSOD1b), while the elution profile of
CLSOD2 revealed the presence of one peak of the enzyme
activity. After the chromatography on the sephacryl S-300
column, the specific activity of CLSOD1a was increased to
2775.21 units / mg protein which represents 12.96-fold
- 226 - Summary
purification over the n-butanol extract with 13.2 % yield and
the specific activity of CLSOD1b was increased to 3705.70
units / mg protein which represent 17.30-fold purification over
the n-butanol extract with 23.2 % yield. Also, the specific
activity of CLSOD2 was increased to 2880.217 units / mg
protein which represent 13.45-fold purification over the nbutanol
extract with 17.2 % yield. By gel filtration, the
molecular weight of the native form of the three isoenzymes
CLSOD1a, CLSOD1a and CLSOD2 were calculated to be 120
± 1.3 kDa, 38 ± 1.8 kDa and 68 ± 1.3 kDa respectively. All
isoenzymes CLSOD1a, CLSOD1a and CLSOD2 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 CLSOD1a, CLSOD1a and CLSOD2
coincided with their enzyme activity band confirming that the
single protein band is the enzyme band. By SDS-PAGE, the
subunits molecular weight of CLSOD1a, CLSOD1a and
CLSOD2 was estimated to be 59 ± 2.9 kDa, 37 ± 1.6 kDa and
65 ± 2.4 kDa respectively. These results indicate that
CLSOD1b and CLSOD2 are monomeric proteins composed of
one subunit, while CLSOD1a is homodiameric protein
composed of two identical subunits. By isoelectrofocusing,
CLSOD1a, CLSOD1b and CLSOD2 showed a single
molecular species with an isoelectric point (pI) value of 7.5-
- 227 - Summary
7.7 for CLSOD1a, 7.8-8.0 for CLSOD1b and 5.6-5.8 for
CLSOD2. CLSOD1a, CLSOD1b and CLSOD2 displayed their
optimum activity at pH 7.8, 7.6 and 7.5 respectively. The
activity of the CLSOD1a isoenzyme was increased about 1.13,
1.19, 1.20 and 1.30 fold in the presence of 2 mM NiCl2, CoCl2,
ZnCl2 and CuCl2, and increased about 1.26, 1.44, 1.88 and
2.22 fold in the presence of 5 mM NiCl2, CoCl2, ZnCl2 and
CuCl2. In contrast, MgCl2 and FeCl2 were found to be potent
inhibitors of CLSOD1a, while CaCl2 was a moderate inhibitor
of CLSOD1a. The activity of the CLSOD1b was increased
1.13, 1.15, 1.21, 1.23 and 1.3 fold in the presence of 5 mM
ZnCl2, NiCl2, CoCl2, CaCl2 and CuCl2 respectively. In contrast,
MgCl2 and FeCl2 were found to be potent inhibitors of
CLSOD1b. The activity of the CLSOD2 was increased 1.15
and 1.153 fold in the presence of 2 mM CaCl2 and MnCl2 and
increased 1.11 and 1.36 fold in the presence of 5 mM MgCl2
and MnCl2. In contrast, CoCl2, NiCl2 and FeCl2 were found to
be moderate inhibitors of CLSOD2. The activity of the
CLSOD1a, CLSOD1b and CLSOD2 are visualized on 7 %
native PAGE in the presence of 5 mM CuCl2, ZnCl2 and
MnCl2. The presence of CuCl2 and ZnCl2 increased the
CLSOD1a and CLSOD1b activity band and did not affect the
CLSOD2, while MnCl2 obviously increased the CLSOD2
activity band. Potassium cyanide is found to be the most potent
- 228 - Summary
inhibitor of the CLSOD1a and CLSOD1b. Sodium dodecyl
sulphate is found to be a potent inhibitor of the activity of
CLSOD2. EDTA, DL-dithiothreitol, -Mercaptoethanol and 1,
10 phenanthroline inhibited the CLSOD1a, CLSOD1b and
CLSOD2. PMSF inhibited CLSOD1b and iodoacetamide
inhibited CLSOD2. Potassium dichromate is found to be a
potent inhibitor of CLSOD1a, CLSOD1b and CLSOD2. On
7% native PAGE; CLSOD1a and CLSOD1b activity band
disappeared in the presence of 5 mM KCN, while CLSOD2
activity band slightly affected. In the presence of 5 mM H2O2,
CLSOD1a activity band less affected and CLSOD1b activity
band disappeared, while CLSOD2 activity band slightly
decreased. The presence of 5 mM NaN3 lowered the intensity
of the CLSOD1a, CLSOD1b and CLSOD2 activity band. The
presence of 5 mM SDS decreased the intensity of the
CLSOD1a and CLSOD1b activity band slightly and the
intensity of the CLSOD2 activity band completely.
3- Sheep liver superoxide dismutase isoenzymes
The starting specific activity in the n-butanol extract was
found to be 116.963 unit / mg protein. Most of the superoxide
dismutase activity was precipitated with 1 volume of
prechilled acetone. More than 88.4 % of the superoxide
dismutase activity was recovered in the acetone fraction and
- 229 - Summary
the specific activity of the enzyme was increased more than
1.5-fold. Two major peaks exhibited the superoxide dismutase
activity were resolved on DEAE-cellulose column and eluted
with 0.05 M and 0.2 M NaCl and designated SLSOD1 and
SLSOD2. The Sheep liver superoxide dismutase specific
activity of the pooled fractions of the two peaks SLSOD1 and
SLSOD2 were increased 4.15 and 2.41 fold over the n-butanol
extract with recovery of 38.0 % and 19.0 % respectively. After
the chromatography on the sephacryl S-300 column, the
specific activity of SLSOD1 was increased to 2500.12 units /
mg protein which represent 21.47-fold purification over the nbutanol
extract with 23.9 % yield. Also, the specific activity of
SLSOD2 was increased to 2243.78 units / mg protein which
represent 19.27-fold purification over the n-butanol extract
with 16.9 % yield. By gel filtration, the molecular weight of
the native form of SLSOD1 and SLSOD2 were calculated to
be 57 ± 2.7 kDa and 66 ± 2.9 kDa respectively. Both SLSOD1
and SLSOD2 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 SLSOD1 and SLSOD2 coincided with their
enzyme activity band confirming that the single protein band is
the enzyme band. By SDS-PAGE, the subunits molecular
weight of SLSOD1 and SLSOD2 was estimated to be 58 ± 2.3
- 230 - Summary
kDa and 67 ± 2.8 kDa respectively. These results indicate that
both SLSOD1 and SLSOD2 are monomeric proteins
composed of one subunit. By isoelectrofocusing, both
SLSOD1 and SLSOD2 showed a single molecular species
with an isoelectric point (pI) value of 6.9-7.1 for SLSOD1 and
5.6-5.8 for SLSOD2. Both SLSOD1 and SLSOD2 displayed
an optimum activity at pH 7.8 and pH 7.6 respectively. The
activity of the SLSOD1 is increased about 1.10, 1.16, 1.21,
1.22 and 1.44 fold in the presence of 2 mM CaCl2, ZnCl2,
NiCl2, CoCl2, and CuCl2 respectively, and increased about 1.19,
1.27, 1.29 and 1.92 fold in the presence of 5 mM CaCl2, CoCl2,
ZnCl2 and CuCl2 respectively. In contrast, MgCl2 was found to
be a moderate inhibitor of SLSOD1 while FeCl2 was a potent
inhibitor. The activity of the SLSOD2 was increased about
1.10 and 1.36 fold in the presence of 2 mM MgCl2 and MnCl2,
and increased about 1.14 and 1.65 fold in the presence of 5
mM of MgCl2 and MnCl2. In contrast, CoCl2, NiCl2 and FeCl2
were found to be moderate inhibitors of SLSOD2. The activity
of both SLSOD1 and SLSOD2 are visualized on 7 % native
PAGE in the presence of 5 mM CuCl2, ZnCl2 and MnCl2. The
presence of CuCl2 and ZnCl2 increased the SLSOD1 activity
band and didn’t affect the SLSOD2, while MnCl2 obviously
increased the SLSOD2 activity band. Potassium cyanide is
found to be the most potent inhibitor of the activity of
- 231 - Summary
SLSOD1. Sodium dodecyl sulphate is found to be a potent
inhibitor of the activity of SLSOD2. EDTA, DL-dithiothreitol,
-Mercaptoethanol and 1, 10 phenanthroline inhibited both
SLSOD1 and SLSOD2. PMSF inhibited SLSOD1 and
iodoacetamide inhibited SLSOD2. Potassium dichromate is
found to be a potent inhibitor of both SLSOD1 and SLSOD2.
On 7 % native PAGE; SLSOD1 activity band disappeared in
the presence of 5 mM KCN, while SLSOD2 activity band
more or less remained similar to the control band. In the
presence of 5 mM H2O2, SLSOD1 activity band disappeared,
while SLSOD2 activity band more or less remained similar to
the control band. 5 mM NaN3 lowered the intensity of the
SLSOD1 activity band slightly and the intensity of the
SLSOD2 activity band sharply. The presence of 5 mM SDS
decreased the intensity of the SLSOD1 activity band slightly
and the intensity of the SLSOD2 activity band completely