can you describe please the contents of a simple reaction to measure catalase activity in a plant extract and is it possible to be a water extract or should it be in phosphate buffer? thanks a lot
The fresh root samples of 0.5 g were grinded and homogenized with 5.0 mL extraction buffer containing 50 mM phosphate buffered potassium (PBP, pH7.0), 0.4% polyvinylpoly pyrrolidone (PVP) in a mortar and pestle on the ice. The homogenate was centrifuged at 10, 000 × g for 30 minutes to produce supernatant. All operations were carried out at 4°C. For CAT activity, the reaction mixture contains 1.9 mL of 100 mM PBP (pH 7.0), 0.1 mL of enzyme extract, and 1.0 mL of 0.075% H2O2 solution. CAT activity was assayed in 1.0 minute at 240 nm
The fresh root samples of 0.5 g were grinded and homogenized with 5.0 mL extraction buffer containing 50 mM phosphate buffered potassium (PBP, pH7.0), 0.4% polyvinylpoly pyrrolidone (PVP) in a mortar and pestle on the ice. The homogenate was centrifuged at 10, 000 × g for 30 minutes to produce supernatant. All operations were carried out at 4°C. For CAT activity, the reaction mixture contains 1.9 mL of 100 mM PBP (pH 7.0), 0.1 mL of enzyme extract, and 1.0 mL of 0.075% H2O2 solution. CAT activity was assayed in 1.0 minute at 240 nm
Preperation
Tissues were homogenized in the ice cold 10 volume of isotonic buffer in a glass homogenizing tube. The homogenate was centrifuged for 5-10 min at 700g to remove the debris. Catalase activity of tissue samples is expressed on a milligram wet weight or milligram total N basis.
Reagents
Ø 0.01M phosphate buffer, pH 7.0
Solution A – 0.01M Sodium dihydrogen phosphate solution
Solution B – 0.01M Disodium hydrogen phosphate solution
Mix 39 ml of solution A and 61 ml of solution B and make up the total volume to 100 ml with distilled water.
Ø Stock dichromate-acetic acid solution: Mixed a 5% potassium dichromate was mixed with glacial acetic acid (1:3 ratio).
Ø Working dichromate/ acetic acid solution: The stock solution was diluted in 1:5 ratio with water to make the working dichromate/ acetic acid solution.
Procedure
The assay mixture contained 0.5 ml of 0.2 M H2O2, 1 ml of phosphate buffer and 0.4 ml of water. To initiate the reaction 0.2 ml of tissue homogenate / 0.2 ml of serum was added. 2 ml of the dichromate/acetic acid reagent was added after 0, 30, 60 and 90 seconds of incubation. To the control tube the enzyme was added after the addition of the acid reagent. The test tubes were kept in boiling water bath (600C) for 10 min and then cooled. The color developed was read at 620 nm against a reagent blank in UV-visible spectrophotometer. Standards of H2O2 in the range of 2-10 μM were taken and preceded as test with blank containing reagent alone. The activity of catalase was expressed as μmole of H2O2 consumed/ min/ mg protein.
CATALASE
Catalase activity was assayed by estimating the initial rate of disappearance of hydrogen peroxide by the method of Chance and Maehly, (1985) and they forming titanium hydro peroxide complex (Teranishi et al., 1974; Prochazskova et al., 2001).
The known amount of tissue was homogenized in 0.1 mM phosphate buffer (pH 7.0), 6mM H202 and 0.2ml enzyme extract. The reaction was stopped after 5 minutes by the addition of 2ml of titanium reagent, which also formed yellow titanium-hydrogen peroxide. After 30 minutes aliquot was centrifuged at 10,000g for 10 minutes. Absorbance of supernatant was recorded at 410nm wavelength using spectrophotometer.
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