Experiments for the determination of peroxidase activity in plants

Summary

Peroxidase is commonly found in plant tissues, and its activity is related to the metabolic strength and cold and disease resistance of plants, which regulates the level of IAA in metabolism and acts as a kind of reactive oxygen defense substance, eliminating the toxic effects of H2O2 produced in the organism. Therefore, it is often measured in scientific research.

Operation method

Experiments for the determination of peroxidase activity in plants

Principle

In the presence of hydrogen peroxide, peroxidase can oxidize guaiacol to produce tea-colored 4-o-methoxyphenol, and the activity of the enzyme can be found by measuring the absorbance (A) value of the product at 470 nm.

Materials and Instruments

Plant leaves
Phosphate buffer Guaiacol
Spectrophotometers Centrifuges Tubes Mantles Pipettes Pipette racks Test tubes Test tube racks Earwash balls

Move

I. Materials, instrumentation and reagents

1. Materials: plant leaves

2. Instrumentation: spectrophotometer, centrifuge, centrifuge tube, mortar and pestle, pipette, pipette rack, test tube, test tube rack, ear washing ball.

3. reagents and preparation:

0.1 mol-L-1 phosphate buffer (pH 7).

Reaction solution (100 ml of 0.1 mol-L-1 phosphate buffer (pH 6) with 0.5 ml guaiacol, 1 ml 30 % H2O2, shake well).

II. Experimental steps

1. Enzyme extraction

Weigh 1g of plant leaves, cut them into pieces and place them in a frozen mortar, add a small amount of quartz sand and add a total of 10 ml of enzyme solution in two additions.

Add a small amount of quartz sand, add a total of 10 ml of pH 7 phosphate buffer in two times, grind it into a homogenate, pour it into a centrifuge tube, centrifuge it at 8000 r/min for 15 min, the supernatant is the crude enzyme extract, and pour it into a small test tube to be placed in a low-temperature standby.

2. Determination of enzyme activity

Pipette 3 ml of reaction solution in a test tube, add 0.02 ml of enzyme extract (depending on the enzyme activity can increase or decrease the amount of addition), quickly shaken well and poured into the optical diameter of 1cm colorimetric cup, with the reaction solution without enzyme solution as a blank control, at the wavelength of 470nm, time scanning mode, to determine the change in absorbance value in 3min, take the linear changes in the portion of the change in absorbance per minute to calculate the change in absorbance values (△ A470).

Calculation of enzyme activity

Calculate the relative activity of the enzyme according to the following formula

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