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.