H2o2 Reaction Lab Report

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2.9. Estimation of Hydrogen peroxide (H2O2)
213 The concentration of H2O2 was determined by the method of Okuda et al (38). Fresh leaf
214 sample (0.5 g) was grounded in ice-cold 200 mM HClO4 and was then centrifuged at
215 1200 g for 10 min followed by neutralization of HClO4 of the supernatant with 4M KOH.
216 The insoluble KClO4 was eliminated by further centrifugation at 500g for 3 min. In a
217 total volume of 1.5 mL, the reaction mixture contained 1 mL of the eluate, 400 mL of
218 12.5 mM 3-(dimethylamino) benzoic acid in 0.375 M phosphate buffer (pH 6.5), 80 mL
219 of 3-methyl-2-benzothiazoline hydrazone and 20 mL of peroxidase (0.25 unit). The
220 reaction was started by the addition of peroxidase and the increase in absorbance was
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For ascorbate peroxidase assay extraction
241 buffer was supplemented with 1.0 mM ascorbic acid. The homogenate was centrifuged at
242 15,000×g for 15 min at 40C, and the supernatant was used as a crude enzyme extract.
243 Spectrophotometric determinations were performed using UV visible spectrophotometer
244 (UV-1700, Shimadju, Japan).
245 2.11.2. Estimation of superoxide dismutase (SOD) activity
246 SOD activity was estimated by its ability to catalyse NBT to formazan at 560nm
247 according to the method of Beyer and Fridovich (40). Five ml of reaction mixture
248 containing 50 mm phosphate buffer (pH 7.8), 13 mm methionine, 75 mm NBT, 2 mm
249 riboflavin, 0.1 mm EDTA and the enzyme extract. Absorbance of sample was read at 560
250 nm. The difference of percentage reduction of colour development in blank and the
251 sample was calculated. Fifty percent reduction in the colour was taken as one unit of
252 enzyme activity and was expressed in enzyme units per milligram protein (U mg-1
253 protein).
254 2.11.3. Catalase (CAT) activity
255 With slight modifications, catalase activity was assayed according to the method of Aebi
256 (41) by monitoring the disappearance of H2O2 at 240 nm. The 3 ml reaction

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