Dear Mohammed

You can see below a method for measuring catalase (CAT) activity:

The plant sample is ground at 4 oC in a porcelain mortar with homogenization buffer containing 100 mM K2HPO4 pH 7.0, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 0.3 % ethanol. The homogenization is carried out using 5 ml buffer per g fresh sample weight. The homogenate is centrifuged at 3,300 g for 5 min, at 4 oC. The resulting supernatant is subsequently used for both CAT activity and protein concentration estimation. CAT is assayed by its depletion of H2O2, followed at 240 nm (Blum and Fridovich, 1983).

The CAT assay consists of mixing (at 30 oC) 0.9 ml sample (proper dilution of the homogenate supernatant) and 0.1 ml 0.09 M H2O2 stock solution (made fresh in homogenization buffer). The linear absorbance decrease (vs time) of the assay mixture is measured at 240 nm in a spectrophotometer. The absorbance decrease rate of consumed H2O2 is converted to CAT units (U) from a pure CAT standard curve.

Dear Mohammed,

There a number of reliable spectrophotometric protocols to determine (i) activities of antioxidant enzymes such as catalase, peroxidases (guaicol peroxidase, ascorbate peroxidase), superoxide dismutase, monodehydroacorbate reductase, degydroascorbate reductase and glutathione reductase; and (ii) non-enzymatic antioxidants such as ascorbate, glutathione, phenols, carotenoids, proline etc.

You should be clear with what exactly you are looking for and why? For preparing crude enzyme extract, buffer composition will depend on the plant species you are using. I shall not advise you to use PMSF unless and until your plant or its parts possess high protease activity. However, extraction buffer should have (i) EDTA; (ii) cysteine or dithiotreitol or beta-mercaptoethanol;  and (iii) PVP or PVPP (if your plant or part that you are using contain phenols).

You may download two or more of our papers where we dealt with estimation of enzymatic and non-enzymatic antioxidants.

Prasad et al. 1999; Alia et al. 1999; Shabnam et al. 2014

I agree with the suggestions of Prof. Pardha. In my opinion, potassium-phosphate buffer pH 7.5 containing PVP, EDTA and Triton-X-100 will extract all the antioxidative enzymes. Remember to homogenize your samples at 4 degree Celsius. This will prevent your enzymes from degradation. Better you store your enzyme extract at -20 degree celsius till assay is done. If you need further help consult my papers available at researchgate. Hope this will help.

Amit.

anti oxidant enzyme (catalase) can be assayed using aebi method (pH=7 and H2O2) its easy and chip , samples must be homogenized using a homogenizer or blinder in the assay buffer , then you can estimate enzyme activity.

FOR CATALASE,

You can use a colorimetric method described by Sinha, 1972. The reaction mixture will containe 0.01 M phosphate buffer pH 7.0 (5 ml), tissue homogenate (1 ml) and 2 M H2O2 (4 ml). The reaction was stopped by the addition of 2 ml dichromate-acetic acid reagent (5% potassium dichromate and glacial acetic acid mixed in 1:3 ratios), at 0, 1, 2, and 3 minutes, followed by heating in boiling water for 10 minutes, and then cooled at room temperature. The absorbance was read at 570 nm.

Superoxide dismutase (SOD) will be determined by the method of Misra and Fridovich, 1972. The method is based on the ability of superoxide dismutase to inhibit autooxidation of adrenaline to adrenochrome at alkaline pH. The unit of enzyme activity is defined as the enzyme required for 50% inhibition of adrenaline auto-oxidation. the reaction mixture contains 2.5 ml of bicarbonate buffer pH 10.2, 0.3 ml of adrenaline, and 0.2 ml of sample. absorbance will be read at 480 nm.