Determination of the rate of oxygen radical production
Understand the principles and methods of hydroxylamine oxidation for the determination of oxygen radicals.
Principle
The basic principle of the determination of the rate of generation of oxygen radicals is to use hydroxylamine oxidation to detect the O2-content in biological systems. 0/ reacts with hydroxylamine to form NO; ,N()r under the action of p-aminobenzenesulfonic acid and W-congestamine to form a pink azo dye, which absorbs significantly at 530 nm, and the absorbance value can be used to calculate the amount of 0/ in the sample. Based on the measured absorbance value, the standard curve of NO was checked and the absorbance value was converted to ［NO；］, and then according to the reaction formula of hydroxylamine and 0；・ (as follows), the stoichiometry of ［0；・］ from ［NOF］ to ［0；・］ was used to obtain ［O；・］. o Based on the recording of the time of the reaction between the sample and the hydroxylamine and the fresh weight of the sample, the rate of production of ［O；・, Cptmol - ( min - mgFW), was calculated. min - mgFW).
NH.OH + 2O;- +undefined- NO7+H2O2 + H2O
Operation method
Determination of the rate of oxygen radical production
Principle
The principle of the oxygen radical generation rate measurement is to use the hydroxylamine oxidation method to detect the O2-content in biological systems. 0/ reacts with hydroxylamine to form NO; ,N()r under the action of p-aminobenzenesulfonic acid and W-congestamine to form a pink azo dye, which is absorbed at 530 nm, and the absorbance value is used to calculate the amount of 0/ in the sample. Based on the measured absorbance value, the standard curve of NO; was checked, and the absorbance value was converted to [NO;], then according to the reaction formula of hydroxylamine and 0;・ (as follows), the stoichiometry of [0;・] from [NOF] was carried out, and ［O；・］ was obtained. o Based on the recorded time of the reaction between the sample and hydroxylamine and the fresh weight of the sample, the rate of production of O;・ was obtained, which was calculated as Cptmol -（ min - mgFW）. NH.OH + 2O;- +undefined- NO7+H2O2+ H2O
Materials and Instruments
Material: Young and senescent plant leaves or other plant tissues subjected to adversity stress, respectively. Move The basic procedure for the determination of the rate of oxygen radical production can be divided into the following steps:1. Preparation of nitrite standard curve: Prepare 2 mL each of 0, 5, 10, 15, 20, 25 and 30" nol-L_1 NaNO2 with 100 Mmol-L_1 NaNO2 (7 mg -) mother liquor, add 2 mL of p-aminobenzenesulphonic acid and 2 mL of cetrimide respectively, and keep warm for 20 min at 25 ℃, and then determine the OD 彻, and plot the OD value against "NO -Then the OD was measured, and the standard curve of nitrite was made by plotting the absorbance value at 530 nm with "NO2. Preparation of plant extract: Take 2 g of the above plant tissue, add 50 mmol - L 1 phosphate buffer (pH 7.8) 2 mL, grind it thoroughly, centrifuge it at 10 000 r - mirT for 20 min, and then condense the supernatant to 3 mL. This solution is O; ・The solution to be tested is produced.3. Measurement of production rate /Determination of production rate: Add 0.5 mL of 50 mmol・L'1 phosphate buffer (pH 7.8) and 1.5 mL of 1 mmol-17 1 hydroxylamine hydrochloride to 0.5 mL of sample extract, shake well, and keep warm at 25°C for 1 h. Then add 2 mL of 17 mmol-L "p-aminobenzenesulfonic acid (prepared by glacial acetic acid: water=3:1) and 2 mL of 7 mmol-L 1 a-turbinamide (prepared by glacial acetic acid: water=3:1) and 2 mL of 7 mmol-L 1 a-turbinamide (prepared by glacial acetic acid: water=3:1) to the extract, shake well. L 1 a-turbinamide (prepared by glacial acetic acid: water=3:1), mixed, and kept at 25°C for 20 min, and then the absorbance value at 530 nm was measured by spectrophotometer.4. Calculation of results: The rate of () broad production in plant tissues of different treatments was calculated according to the reaction equation in the experimental principle, namely宀,...., ln cXV.XNO2 - production rate term mol ・(min - gFW) ']= 2X 嵐 7 -Where: c a concentration of NO in the sample found on the standard curve (mol・L);N a dilution of the sample extract. i.e. 4;N a dilution times of sample extract. i.e. 4; 이이엝 丄, i.e. 3X10-3 L.The amount of 2-0/ is 2 times that of NO;W - fresh weight of plant tissue, g;W - fresh weight of plant tissue, g; T - reaction time, i.e. 60 min. Caveat If the sample contains a large amount of chlorophyll that will interfere with the determination, chlorophyll can be extracted by adding an equal volume of ethanol after a warm bath of the sample with hydroxylamine, followed by the addition of p-aminobenzenesulfonic acid and a-chloramine for NO2and a-chloramine for the color development reaction of NO For more product details, please visit Aladdin Scientific website.
Materials: mortar, balance, thermostatic water bath, graduated test tubes, volumetric flasks, measuring cylinders, centrifuge, spectrophotometer.
Reagents:
①series of concentrations of NaNO
2
②17 mmol・L
-②17 mmol・L
p-aminobenzenesulfonic acid (2.944 g・L-1)
-1
(2.944 g ・L-1 , prepared with glacial acetic acid: water = 3 : 1)
③7 mmol・L
③7 mmol・L-1
α-congestamine (1.0 g・ L-1 , prepared with glacial acetic acid: water = 3 ： 1)
L-1 , prepared with glacial acetic acid: water = 3 : 1)
(1.0 g・ L-1 , prepared by glacial acetic acid: water = 3 : 1)
④50 mmol-L
④50 mmol-L
④50 mmol-L-1 phosphate buffer
⑤1 mmol・L-1 , prepared with glacial acetic acid: water = 3:1
⑤1 mmol ・ L-1 phosphate buffer ⑤1 mmol ・ L-1
Hydroxylamine hydrochloride (70 mg・L)
-1
(70 mg ・ L -1)