Glutathione Peroxidase Lab Report

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4.2.3.5. Antioxidant assays in gastric tissue homogenate
4.2.3.5.1. Catalase activity
Catalase activity in the gastric tissue was measured according to the procedures of Sinha (1972). One ml of homogenate solution was taken and mixed with 5 ml of phosphate buffer of pH 7.4. To this 4 ml of 0.2 M H2O2 was added and time noted. Exactly after 180 seconds of adding H2O2, a set of 1ml of reaction mixture from the above was taken and 2 ml dichromate acetic acid was added. It was then kept in a boiling water bath for 10 minutes. All the tubes were then cooled under running tap water and finally readings were taken at 570 nm against reagent blank. The catalase activity in gastric tissue was expressed as µmoles H2O2 consumed /mg protein /min. 4.2.3.5.2. Glutathione peroxidase activity
Estimation of glutathione peroxidase was made according to the methodology of Flohe and Gunzler, 1984. Took 0.2 ml of EDTA, sodium azide, reduced glutathione and H2O2 in a test tube. Then added 0.4 ml of Sodium Phosphate buffer of pH 7 and 0.2 ml of
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(1979). Pipetted out 0.1ml of gastric tissue homogenate, 0.2 ml of 8.1% sodium dodecyl sulfate (SDS), 1.5 ml of 20% acetic acid solution and 1.5 ml of 0.8% aqueous solution of TBA into suitably labeled test tubes. The reaction mixture was then made up to 4.0 ml with distilled water and heated at 95°C for 60 min in water bath. After cooling the test tubes under tap water, 1.0 ml of distilled water and 5.0 ml of the mixture of n-butanol and pyridine (15: 1, v/v) was added and shaken vigorously. Centrifuged the test tubes at 4000 rpm for 10 minutes and the absorbance of the upper layer were measured at 532 nm. Standard malondialdehyde was taken as the standard and was processed in the same manner. The level of lipid peroxide was expressed as µmoles of MDA formed /g wet

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