Activities assay of SOD, catalase, GPx and LDH activities by gel
zymogram
Super oxide dismutase. Three different SOD activities were
assayed, first in breast tumor and corresponding surrounding tissue,
second in the serum of animal model treated with DAS, Chalcone and
DAS+Chalcone for 7 days, 24, 48 and 72 hours and thirdly in the in vitro
treated heapatocytes (3, 5 and 12 hour). A tablet of nitro blue
tetrazolium (NBT) dissolved in 30 ml of water and the non-denaturing
(10%) acrylamide gel kept soaked in it for 30 minfollowed by 40 ml SOD
solution containing 0.028 M tetramethylethylenediamine (TEMED), 2.8 x
10-5 M riboflavin, and 0.036 M potassium phosphate at pH 7.8) for 15
minin shaking condition. Finally the gel was placed on clean acetate
sheet and illuminated for 5 to 15 min and thenunder a UV illuminator
(23)
Catalase. A non-denaturing gel (8%) was loaded with a 25µg of
cytosol (from in-vitro treated rat hepatocytes for 3, 5 and 12 hours
with DAS, Chalcone and DAS+Chalcone) and electrophoresed for 3 h at 40
mA at 4 °C. The gel was washed 3 × 10 min in distilled water and
incubated in 0.003% H2O2 for 10
minutes, followed by staining with 2% ferric chloride and 2% potassium
ferricyanide. In this critical step two reagents were not mixed prior to
staining rather poured together directly on top of the gel. The gel
became greenish blue except in the position containing the catalase.
After rinsingwith distilled water the gel was scanned when the maximum
contrast between the band and the background was obtained (23).
Glutathione peroxidase. A non-denaturing gel (8%) was loaded
with a 150 µg of protein obtained from rat hepatocytes treated for
different times with DAS and/or Chalcone. Electrophoresis carried out
for 3 h at 40 mA at 4°C. The gel was washed 3 × 10 min in distilled
water containing 1 mM GSH and incubatedin 0.008% cumene hydroperoxide
for 10 minutes. Then 1% ferric chloride (w/v) containing 1% GSH and
1% potassium ferricyanide (w/v) containing 1% GSH were prepared
separately and poured together directly on the gel. When achromatic
bands begin to form (5 – 15 min), the stain was poured off and the gel
was washed extensively with distilled water. The image of the bands
demonstrated GPx activity was evaluated. The cumene hydroperoxides
instead of H2O2 performed as a good
substrate and the GPx (contains a selenium in the active site) can
utilize this hydroperoxide to determine total peroxidase activity
according to the established protocol as described in the referenced
article (23).
Lactate dehydrogenase (LDH). LDH zymogram in tumour and its
corresponding surrounding tissue obtained from breast cancer
pateints,was performed by using a standard protocol (24) with slight
modification. LDH zymograms were obtained by separating 10 mg of protein
on a native PAGE gel of 8 % for 2 h at 4 W, then soaked in water
followed by washing twice with water. The LDH activity was detected by
soaking the gel in a staining solution containing 0.1 M Tris-HCl (pH
8.0), 750 mM NAD1, 200 mM lithium lactate, 0.1 mg of NBT - nitroblue
tetrazolium per ml, and 0.02 mg of phenazine methylsulfate per ml.
Enzyme activity bands were developed from the conversion of NAD1 to
NADH, which were clearly visible. The gel was scanned under the gel doc
system.