Determination of nitrate reductase activity

NO3- taken up by plant roots must be metabolically reduced before it can be further utilized by the plant. Nitrate reductase (EC.1.6.6.1, abbreviated as NR) is the first enzyme in nitrate assimilation and the rate-limiting enzyme, which is in the key position of nitrogen metabolism in plants.NR is related to the uptake and utilization of nitrogen fertilizers by plants, and it has an important influence on the yield and quality of crops, therefore, the activity of nitrate reductase has been regarded as one of the indexes of plant nutrition or fertilizer application on the farmland, and also one of the indexes of varietal selection and breeding. Through this experiment, we will initially master the principle and method of determining nitrate reductase activity and understand the characteristics of nitrate reductase.

Principle

The basic principle of nitrate reductase activity measurement is that nitrate reductase catalyzes the reduction of nitrate to nitrite in the plant body, and the ^NO2- produced can penetrate from plant tissues into the external solution and accumulate in the solution, and the increase in the ^NO2-content in the reaction solution is measured, which indicates the size of the enzyme activity.

Under certain conditions, the amount of ^NO2- generation and nitrate reductase activity is positively correlated. ^NO2- content can be measured by sulfonamide colorimetric method, i.e., under acidic conditions and p-aminobenzenesulfonic acid (or p-aminobenzenesulfonamide) diazotization reaction occurs, the resulting diazo compounds and ^{a-naphthylamine} (or N-naphthylethylenediamine hydrochloride) to generate red azo compounds can be colorimetric determination at 540 nm. The reaction is as follows:

Nitrate reductase activity is generally measured in vivo or in vitro. In the in vitro method, the material is ground into a homogenate, the residue is removed by filtration or centrifugation, and the supernatant is used as the crude nitrate reductase solution for the assay. Due to the loss of NADH during grinding, additional NADH is necessary for the determination. In vivo method is directly measured with fresh tissue, ^NO3- in the environment into the cell, the nitrate reductase is reduced to ^{NO2- and} diffuse to the outside of the cell in the solution accumulation, the measurement of ^NO2-content in the solution can be known to the size of the nitrate reductase activity. The in vivo method does not destroy the original enzyme reaction system of the cell, and NADH can be generated continuously by the metabolic reaction without addition. The in vivo method is simple, rapid, and does not require expensive equipment and low temperature conditions, but the repeatability is not good, and a certain amount of repetition should be done. In this experiment, the in vivo method was used to determine the nitrate reductase activity.

Operation method

Determination of nitrate reductase activity

Principle

The basic principle of nitrate reductase activity is that nitrate reductase catalyzes the reduction of nitrate to nitrite in plants, and the NO2- generated can penetrate from plant tissues to the external solution and accumulate in the solution, and the increase of NO2-content in the reaction solution is measured, that is, it shows the size of enzyme activity. Under certain conditions, the amount of NO2- generation and nitrate reductase activity is positively correlated. NO2-content can be measured by sulfanilamide colorimetric method, that is, under acidic conditions and p-aminobenzenesulfonic acid (or p-aminobenzenesulfonamide) to occur diazotization reaction, the generated diazo compounds and a-naphthalene amine (or N-naphthalene ethylenediamine hydrochloride) to generate red azo compounds can be colorimetric measurement at 540 nm. The reaction is as follows: Nitrate reductase activity is generally measured in vivo or in vitro. In the in vitro method, the material is ground into a homogenate, the residue is removed by filtration or centrifugation, and the supernatant is used as the crude nitrate reductase solution for the assay. Due to the loss of NADH during grinding, additional NADH is necessary for the determination. In vivo method is directly measured with fresh tissue, the NO3- in the environment into the cell, the nitrate reductase is reduced to NO2- and diffuse to the outside of the cell in the solution accumulation, the determination of the NO2-content in the solution can be measured to know the size of the nitrate reductase activity. The in vivo method does not destroy the original enzyme reaction system of the cell, and NADH can be continuously generated by the metabolic reaction without addition. The in vivo method is simple, rapid, and does not require expensive equipment and low-temperature conditions, but the repeatability is not good, and a certain amount of repetition should be done. In this experiment, the in vivo method was used to determine the nitrate reductase activity.

Materials and Instruments

Material: fresh leaves of wheat, corn, cabbage, rape, tobacco and other crops may be used.
Reagents:
① NaNO
2
Standard solution: accurately weigh NaNO
2
0.1000 g, dissolve with water and then volume to 100 mL, absorb 5 mL of this solution, dilute with water and volume to 1 000 mL, i.e., 5 μg-mL-1.
-1
NaNO
2
standard solution.
② 0.1 mol-L
-1
pH 7.5 phosphate buffer:
Liquid A (0.2 mol-L
-1
Na
2
HPO
4
): Weigh the Na
2
HPO
4
-2H
2
O (A.R) 35.61 g, was dissolved in distilled water and was allowed to settle to 1 000 mL;
Liquid B (0.2 mol-L
-1
NaH
2
PO
4
): weigh NaH
2
PO
4
-H
2
O (A.R) 27.6 g, dissolved in distilled water and volume to 1 000 mL. 84.0 mL of liquid A and 16.0 mL of liquid B were mixed well and 100 mL of distilled water was added;
Take 84.0 mL of liquid A and 16.0 mL of liquid B, mix well, and add 100 mL of distilled water. pH was measured with an acidimeter if necessary, and corrected to pH 7.5 with HC1 or NaOH solution.
③ 0.1 mol-L
-1
KNO
3
solution: weigh 2.5275 g KNO
3
(A.R) was dissolved in phosphate buffer solution and was allowed to swell to 250 mL.
④ 1.0% p-aminobenzenesulfonic acid solution: weigh 1 g of p-aminobenzenesulfonic acid, add 25 mL of concentrated hydrochloric acid and volume to 100 mL with water.
⑤ 0.2% a-naphthylamine solution: weigh 0.2 g of a-naphthylamine, add 25 mL of glacial acetic acid, and then volume to 100 mL with water.
Equipment:
① balance
② vacuum pump and vacuum dryer
③ Centrifuge
① Balance ② Vacuum pump and vacuum drier ③ Centrifuge ④ 20 mL graduated test tube
⑤ 50 mL triangular flask
⑥ Water bath
⑦ Thermostat
⑧ Spectrophotometer
⑨ 5 mL, 2 mL, 1 mL pipette

Move

The basic procedure for the determination of nitrate reductase activity can be divided into the following steps. 1:

1. Preparation of standard curve: Take 6 clean test tubes, number them, and add reagents according to the order of Table 7-1.

After shaking well, the sample was kept at 30 ℃ for 20 min, and then the optical density was measured at 520 nm, and the standard curve was plotted with the nitrite content as the horizontal coordinate and the optical density as the vertical coordinate.

2. Determination of enzyme reaction and enzyme activity: Wash the experimental materials (e.g. cabbage leaves) with water, then rinse them with evaporated water, and then absorb them with gauze or filter paper. Cut the materials into 1 cm × 1 cm pieces, mix them well and weigh 4 portions of 1 g each, then put them into 4 50 mL flasks, number them and add various reagents according to Table 7-2.

3. After shaking well, place the triangular flask in a vacuum desiccator and connect the vacuum pump to pump for 10 min. After deflating, the leaves become soft and sink into the solution. Remove the triangular flask into a thermostat, and keep warm at 30 ℃ and dark for 30 min. 4.

4. 2 mL of supernatant was sucked into another test tube, and 4 mL of 1% p-aminobenzenesulfonic acid solution and 4 mL of 0.2% α-chloramine solution were added, and the optical density at 520 nm wavelength was measured at 30 ℃ for 20 min. Subtract the average value of optical density of No.1 and No.2 tubes from the average value of optical density of No.3 and No.4 tubes, and find out the corresponding _NaNO2 content (μg) on the standard curve. 5.

5. Result and calculation: Substitute the _NaNO2 content found on the standard curve into the following formula to calculate the nitrate reductase activity.

NR activity (μg ^NaNO2-g-1 fresh ^weight-h-1 ) = _{NaNO2 (} μg) × total volume of reaction solution/sample weight (g) × time (h)

Caveat

1. vacuum pumping can be carried out in a vacuum thermostat (pumping to a column of 700 mm Hg for 10 min) or in a vacuum desiccator with a vacuum pump connected.

2. Nitrate reductase reaction should be carried out in the dark to prevent the chloroplasts in the photosynthesis of ferric oxide protein reduction NO2- - - - - - - - - - - - - - - - - - - - - - -, to ensure the accuracy of the assay results.

3. Nitrate reductase is an inducible enzyme. Light is one of its inducing conditions, so it should be sampled after photosynthesis has been carried out for a period of time (3 h), and field sampling should be carried out in the morning after 9 o'clock.

4. Inorganic phosphorus promotes the activity of nitrate reductase, so phosphate buffer is commonly used.

5. When preparing the standard solution, add NaNO2When preparing the standard solution, add NaNO 2 standard solution and evaporated water and shake well. For color development, 1% p-aminobenzenesulfonic acid solution should be added and shaken well.

6. The experiment from color development to colorimetry should be consistent, the color development time is too long or too short on the color have an effect.

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