Experimental determination of ascorbic acid oxidase activity in plants

Summary

There are five terminal oxidative enzymes in plants, including ascorbate oxidase, polyphenol oxidase, cytochrome oxidase, flavinase, and alternate oxidase. This complex system of oxidizing enzymes helps the plant to adapt to external environmental conditions.

With different plant species, the oxidizing enzymes are not exactly the same, and the enzyme system changes as the plant grows and develops. For example, rice seeds are dominated by cytochrome oxidase at the beginning of germination, and after 6 to 7 d, the role of ascorbate oxidase dominates. In addition, not only is respiration enhanced after the plant is susceptible to disease, but the oxidizing enzyme system also changes accordingly. For example, terminal oxidases in normal wheat plants are mainly iron-containing enzymes, which are replaced by copper-containing enzymes when leaves are infected with rust.

Thus, the determination of oxidizing enzyme species and activities is of great significance to understand the metabolism of plants and its relationship with environmental conditions. Through the experiment, a simple and rapid principle and method for the determination of ascorbic acid oxidase activity will be mastered.

Operation method

Experimental determination of ascorbic acid oxidase activity in plants

Principle

Ascorbic acid oxidase oxidizes ascorbic acid to dehydroascorbic acid in the presence of oxygen, and at the same time promotes the combination of its hydrogen with oxygen in the air to form water. Ascorbate oxidase is measured by adding a certain amount of substrate (ascorbic acid) and enzyme extract to a reaction flask at the optimum pH and temperature of the enzyme, allowing the enzyme to act for a certain period of time, and then determining the amount of substrate consumed to calculate the enzyme activity. The amount of ascorbic acid consumed can be determined by titrating the remaining ascorbic acid with iodine solution. The reaction formula is as follows: I2 in the above formula comes from the following reaction: KIO3 + 5KI + 6HPO3 --→ 3I2 + 6KPO3 + 3H2O

Materials and Instruments

Rice yellowing seedlings Potato tubers Plant leaves
Phosphate buffer Ascorbic acid Metaphosphoric acid Starch solution Potassium iodate preparation Iodine preparation
Mortar 50 ml measuring cylinder 50 ml triangular flask Microburette Titration rack Pipette Pipette rack Constant temperature water bath Ear washing ball

Move

I. Experimental steps

1. Enzyme extraction

Weigh fresh samples (rice yellowing seedlings 2-3 g, potato tubers with 5 g) cut into pieces and placed in a mortar, add a small amount of quartz sand and pH 6 phosphate buffer, quickly grinding into a homogenate (prior to the buffer cooled with iced water, so as not to make the grinding temperature increase). Wash all the materials with buffer into a 50 ml volumetric flask, fixed volume to the scale. It was placed on a water bath at 18-20°C for 30 min (during which it was shaken several times). The supernatant (enzyme extract) was then filtered through cotton in a clean triangular flask and set aside.

2. Determination of enzyme activity

Take three 50 ml triangular flasks, labeled with a number, and accurately add each test solution according to the following table.


First add 4 ml of pH 6 phosphate buffer, 2 ml of 0.1% ascorbic acid into each bottle, and add 1 ml of 10% trichloroacetic acid into bottle No. 3, then add 3 ml of enzyme solution (depending on the activity of the enzyme, increase or decrease the amount of enzyme) into each bottle every 1 minute, record the time of adding the enzyme solution accurately, and then add 1 ml of 10% trichloroacetic acid into each of the bottles at No.1 and No.2 to terminate the enzyme activity after the enzyme reaction in the water bath at 18-20℃ for 5 min with a shake of the spoon. Immediately after 5 min of enzymatic reaction in 18～20℃ water bath, 1 ml of 10% trichloroacetic acid was added to each bottle of No.1 and No.2 to terminate the enzyme activity, and then 3 drops of 1% starch solution was added to each bottle as indicator, and titration was carried out with mol iodine solution using a micro burette to take the emergence of light orchid color as the end point of the titration. The titration value was recorded.

Common Problems

Calculation of enzyme activity:

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