Determination of catalase (CAT) activity

Summary

When plants age or are exposed to adversity, the metabolism of reactive oxygen species is enhanced in the body resulting in the accumulation of H2O2, which causes cellular damage. Catalase, a pervasive enzyme in all plant tissues, can scavenge H2O2 and is one of the important enzymatic defense systems in plants. Therefore, catalase activity in plant tissues is closely related to the metabolic strength and stress tolerance of plants. Master the principle and method of determining catalase activity by ultraviolet absorption method.

Principle

With strong absorption at 240 nm, 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

Determination of catalase (CAT) activity

Principle

With strong absorption at 240 nm, 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.

Materials and Instruments

Material: plant leaves.
Reagent: 1 mol - H
2
O
2
: 30% H
2
O
O2
is approximately equal to 17.6 mol・L.
Take 30% H
2
O
2
solution 5.68 mL and dilute to 1,000 mL;
0.2 mol - L
-1
pH 7.8 phosphate buffer (containing 1% polyvinylpyrrolidone).
Equipment: UV spectrophotometer, thermostatic water bath, centrifuge, mortar and pestle, 0.5 mL graduated pipette, 10 mL test tube.

Move

The basic procedure for the determination of catalase (CAT) activity can be divided into the following steps:

1. Enzyme extraction: weigh 1.0 g of wheat leaves and add a small amount of phosphate buffer solution of pH 7.8, grind it into homogenate, transfer it to a 10 mL graduated test tube, rinse the mortar with this buffer solution and transfer the rinsing solution to a graduated test tube, fix the volume with the same buffer solution, and centrifuge it for 15 min at 4,000 r - min-1, and the supernatant solution will be the crude extraction solution of catalase.

Determination: Take three 10 mL test tubes, two of which are sample determination tubes and one is a blank tube (the enzyme solution will be boiled to death), and add the reagents according to the order of Table 40-1.

After preheating at 25°C, 0.3 mL of 0.1 mol - L-1H2O2 was added tube by tube , and each tube was timed immediately and poured into a quartz colorimetric cup, the absorbance was measured at 240 nm, and readings were taken at 1 min intervals for a total of 4 min, and then the enzyme activity was calculated after all the three tubes had been measured.

Calculation of the results: The amount of enzyme decreased by 0.1 of A240 in 1 min was regarded as one unit of enzyme activity (U).

The formula: A240=Aso- ( Asi+As2 )/2 (Aso is the absorbance of the control tube with boiled enzyme solution, AS1 and AS2 are the absorbance of the sample tube);

Vt a total volume of crude enzyme extract, mL

V1 - volume of crude enzyme extract, mL.

FW a sample fresh weight, g;

0.1 i A240 Each decrease of 0.1 is 1 enzyme activity unit, U;

t i time from addition of hydrogen peroxide to the last reading, min.

Caveat

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


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Categories: Protocols