Many biotic and abiotic adversities induce the production of hydrogen peroxide (H2O2), and h2o2 plays an important role as a signaling molecule in plant stress tolerance. Therefore, the understanding of the changes of h2o2 in plants can reflect the degree of plant response to adversity. This experiment will help us to understand the principle and method of h2o2 determination.
Principle
The basic principle of the determination of hydrogen peroxide content of H2O2 and titanium sulfate (or titanium chloride) to form a peroxide - titanium complex yellow precipitate, can be dissolved by H2SO4, in the 415 nm wavelength colorimetric determination. Within a certain range, its color shade and H2O2 concentration is linear.
Operation method
Determination of hydrogen peroxide content
Principle
The basic principle of the determination of hydrogen peroxide content is that H2O2 and titanium sulfate (or titanium chloride) form a yellow precipitate of peroxide-titanium complex, which can be dissolved by H2SO4 and measured colorimetrically at 415 nm. Within a certain range, the color shade is linearly related to the concentration of H2O2.
Materials and Instruments
Materials: Young and senescent plant leaves or other plant tissues subjected to adversity. Move The basic procedure for the determination of hydrogen peroxide content can be divided into the following steps:1. Preparation of the standard curve: Take seven 10 mL centrifuge tubes, number them sequentially, and add reagents according to Table 47-1.Table 47-1 Wei.2 Concentration Standard Curve Spiking Table mL （1） Weigh 2~5 g of fresh plant tissues (depending on the amount of H2O2 content), add pre-cooled acetone and a little quartz sand at 4°C into a homogenous mixture in the ratio of 1:1, and then centrifuge for 10 min at 3,000 r min-1, discard the residue, and the supernatant will be the sample extract.(2) Pipette 1 mL of the sample extract, add 5% titanium sulfate and concentrated ammonia according to Table 47-1, and then centrifuge at 3 000 r - min "1 for 10 min after the precipitate is formed, and discard the supernatant. The supernatant was discarded. The precipitate was washed with acetone 3 to 5 times until the phytochrome was removed.(3) Add 5 mL of 2 mol-L '1 sulfuric acid solution to the washed precipitate, allow to dissolve completely, and then determine the volume and colorimetry in the same way as the standard curve.3 . Calculate the result:H2O2 content in plant tissues (卩 mol・g7FW)=^£3Where: 〃 - Amount of H,O2 in the sample found on the standard curve, 卩 mol;K-Total volume of sample extract, mL;V1 -volume of sample extract used in the determination, mL;W - fresh weight of plant tissue・g. Caveat H2O2solutions decompose on their own as they are left to stand, so long-standing H2O2solution should be recalibrated before use. For more product details, please visit Aladdin Scientific website.
Equipments: mortar and pestle, 2 0.2 mL pipettes, 1 5 mL pipette, 7 10 mL volumetric flasks, 8 5 mL centrifuge tubes, centrifuge, spectrophotometer.
Reagents:
①100 μmol・H2O2 solution: take 30% analytically pure H
2
①100 μmol・H2O2 solution: take 30% analytically pure H
H2O2 solution: Take 30% analytical pure H2O2
57 μL, dissolve in 100 mL, and dilute 100 times.
②2 mol・L-l solution: dissolve in 100 mL of 30% analytical pure H2O2 and dilute 100 times.
-②2 mol・L
Sulfuric acid solution
③5% (W/V) titanium sulfate solution
④Acetone
⑤ Concentrated ammonia
After the precipitate was completely dissolved, it was carefully transferred into a 10 mL volumetric flask, and the tube was rinsed with distilled water for a few times, and the washings were combined and concentrated to 10 mL mark, and the color was measured at 415 nm.2. Sample extraction and determination： Reagent Centrifuge tube number 1 2 3 4 5 6 7 100 〃mol ・ L_1 H2O2 solution 0 0.1 0.1 0.2 0.2 0.2 0.4 0.4 0.6 0.8 1.0 Pre-cooled acetone at 4°C 1.0 0.9 0.8 0.6 0.6 0.2 0.2 5% Titanium sulfate solution 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Concentrated ammonia 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Centrifuge at 3 000 rmin-1 for 10 min, discard the supernatant and leave the precipitate. 2 mol - Li sulfuric acid solution 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0