Catalase activity assay experiment

Summary

H2O2 is strongly absorbed at 240 nm, and catalase decomposes hydrogen peroxide, causing the absorbance of the reaction solution (A240) to decrease with reaction time. The activity of catalase can be measured by measuring the rate of change of absorbance

Operation method

Catalase activity assay experiment

Principle

H2O2 is strongly absorbed at 240 nm, and catalase decomposes hydrogen peroxide, causing the absorbance of the reaction solution (A240) to decrease with reaction time. The activity of catalase can be measured by measuring the rate of change of absorbance.

Move

I. Instruments and Appliances

UV spectrophotometer; centrifuge; mortar and pestle; 1 volumetric flask of 250 ml; 2 graduated pipettes of 0.5 ml, 1 graduated pipette of 2 ml; 3 test tubes of 10 ml; constant temperature water bath;

II. Reagents

0.2mol/L pH7.8 phosphate buffer (containing 1% polyvinylpyrrolidone);

0.1mol/L H2O2 (calibrated with 0.1mol/L potassium permanganate).

III. Methods

1. Enzyme extraction: weigh the fresh wheat leaves or other plant tissues 0.5g in a mortar, add 2-3ml 4 ℃ pre-cooled pH7.0 phosphate buffer and a small amount of quartz sand grinding into a homogenate, transferred to a 25ml volumetric flask, and buffer rinse mantle for several times, combined with the rinse solution, and fixed to the scale. Mix well and leave the volumetric flask in the refrigerator at 5℃ for 10min, take the upper part of the clarified solution and centrifuge it at 4000rpm for 15min, and the supernatant is the crude extract of catalase. keep it at 5℃ for spare use.

2. Determination: Take three 10ml test tubes, two of which are sample determination tubes and one is blank tube, add reagents according to the order of Table 40-2.

Ultraviolet absorption method for the determination of _H2O2 sample liquid configuration table

After preheating at 25℃, 0.3ml of 0.1mol/L H2O2 was added tube by tube, and the time was recorded immediately after each tube was added and quickly poured into a quartz colorimetric cup, and the absorbance was measured at 240nm, and the readings were taken once every 1min for a total of 4min, and the enzyme activity was calculated according to the following formula when all the three tubes were measured.

3. Calculation of results:

The amount of enzyme that reduces _A240 by 0.1 in 1 min is taken as 1 unit of enzyme activity (u)

Caveat

Any substance with strong absorption at 240 nm interferes with this experiment.

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