A portion of the oxygen molecules in living organisms, when involved in enzymatic or non-enzymatic reactions, are converted to superoxide anion radicals (O2-) if they accept only one electron.O2- can both interact directly with reactive substances in the body, such as proteins and nucleic acids, and can be derived into H2O2 hydroxyl radicals (-OH) ), monoclinic oxygen (1O2), and others. -OH can trigger the peroxidation of unsaturated fatty acid lipids (RH) to produce a series of free radicals, such as: lipid radicals (-R), lipoxygen radicals (RO-), lipid peroxyl radicals (ROO-) and lipid peroxides (ROOH). Excessive accumulation of free radicals can damage cell membranes and many biomolecules. This experiment focuses on the principle and method of determining oxygen radicals in plants.
Operation method
Experiments for the determination of oxygen radical content in plants
Principle
In living organisms, oxygen acts as an acceptor for electron transfer and generates superoxide anion radicals (O2-) when given a single electron. Using hydroxylamine oxidation, it is possible to determine the amount of O2- in biological systems.O2- reacts with hydroxylamine to form NO2-, which in the presence of p-aminobenzenesulfonic acid and α-naphthylamine produces the pink azo dye (p-Benzenesulfonic acid-azo-α-naphthylamine). The absorbance (A) value was measured at 530nm, and the O2-content in the sample was calculated from the A530 value. The reaction formula is as follows:
Materials and Instruments
Peanuts Mung beans Soybean yellowing seedlings Move I. Material Instrumentation and Reagents 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 ether after a warm bath of the sample solution with hydroxylamine. For more product details, please visit Aladdin Scientific website.
Phosphate buffer Hydroxylamine hydrochloride P-aminobenzenesulfonic acid α-Naphthylamine NaNO2 mother liquor
High-speed freezing centrifuge Spectrophotometer Constant temperature water bath Bowl Test tubes Pipettes Tube racks Pipette racks Earwash balls
1. Materials: hypocotyls of peanut, mung bean, soybean yellowing seedlings and other plant leaves.
2. Instruments and equipment: high-speed freezing centrifuge, spectrophotometer, constant temperature water bath, mortar, test tubes, pipettes, test tube racks, pipette racks, and ear washing balls.
3. Reagent preparation:
50 mmol-L-1 phosphate buffer (pH 7.8)
1 mmol-L-1 hydroxylamine hydrochloride
17 mmol-L-1 p-aminobenzenesulfonic acid (prepared with glacial acetic acid: water = 3:1)
7 mmol-L-1 α-naphthylamine (prepared with glacial acetic acid: water = 3:1)
50 nmol- ml-1NaNO2 mother liquor
Experimental procedure
1. Preparation of extraction solution
Weigh 1g of plant leaves into a mortar and pestle in an ice bath, add 50mmol-L-1 phosphate buffer (pH 7.8) 5 ml, grind into a homogenate, centrifuged at 1000r/min, 4 ℃ for 10min, take the supernatant and then centrifuged at 15,000r/min, 4 ℃ for 20min, the second supernatant is the sample extraction solution.
2. Preparation of nitrite standard curve
2.1 Preparation of series concentration NaNO2 solution
Take 50 nmol- ml-1NaNO2 master solution and dilute it into 0, 10, 20, 30, 40 and 50, nmol- ml-1 standard dilutions.
2.2 Take 7 test tubes, numbered 0 to 6, and add 1 ml of 10, 15, 20, 30, 40, 50, nmol- ml-1NaNO2 standard diluent, respectively, and 1 ml of distilled water to tube 0. Then each tube was further added with 1 ml of 50 mmol-L-1 phosphate buffer, 17 mmol-L -1 p-aminobenzenesulfonic acid 1 ml and 7 mmol-L-1 α-naphthylamine 1 ml, placed in 25 ℃ to develop the color for 20 min, and then the No. 0 tube was used as a blank control, and the absorbance (A) value was measured at 530 nm.
2.3 Standard curve
The standard curve was plotted with the concentration of nitrite (NO2-) in tubes No. 1-6 as the horizontal coordinate and the absorbance value as the vertical coordinate.
3. Determination of O2-content
Take four test tubes, numbered 0 to 3, 1 to the number of tubes were added to the sample extract 0.5 ml (three replicates), 0 tube 0 with distilled water 0.5 ml, then each tube was added to the 50 mmol-L-1 phosphate buffer 0.5 ml, 1 mmol-L-1 hydroxylamine hydrochloride 1 ml, mixed, placed at 25 ℃ for 1 h, and then Then 17 mmol-L-1 p-aminobenzenesulfonic acid 1 ml and 7 mmol-L-1 α-naphthylamine 1 ml were added to each tube, mixed well, and placed at 25 ℃ for 20 min to develop the color, and the absorbance (A) value was measured at 530 nm with No. 0 tube as the blank control.
Calculation of results
According to the measured A530 value, the NO2-concentration in the sample was found from the standard curve, and the NO2-concentration in the sample could be calculated according to the formula (1). Then according to the above reaction between hydroxylamine and O2-, stoichiometry from NO2- to O2-, calculated according to the formula (2), i.e., multiply the NO2-concentration obtained according to the formula (1) by 2 to get the O2-concentration. The O2- production rate can also be obtained by formula (3), based on the reaction time of the sample under test with hydroxylamine and the protein content of the sample. 