Assay of nitrate reductase activity

Nitrate reductase is a key enzyme in the metabolic action of plant nitrogen and is associated with the ability of crops to take up and utilize nitrate nitrogen. It catalyzes the reduction of NO3- to NO2-:

Principle

The basic principle of the assay experiment for the determination of nitrate reductase activity is to

Operation method

Assay of nitrate reductase activity

Principle

Nitrate reductase is a key enzyme in the metabolic action of plant nitrogen and is related to the ability of crops to absorb and utilize nitrate nitrogen. It catalyzes the reduction of NO3- to NO2-: NO3-+NADH+H+→NO2-+NAD++H2O The NO2-created by the reaction can permeate from the tissue to the external solution, and the timing can be determined by measuring the change of NO2-content in a certain reaction solution to know the activity of this enzyme. The change of NO2-content in a certain reaction solution can be measured to understand the size of the enzyme activity.The quantitative determination of NO2-is by the sulfanilamide colorimetric method. This method is simple and sensitive, and can measure changes in NaNO2 content of 0.5 μg/ml.

Materials and Instruments

Phosphate buffer KNO3 solution Sulfanilamide reagent α-naphthylamine reagent NaNO2 standard solution
Model 721 Spectrophotometer Vacuum Pump Thermal Chamber Balance Vacuum Dryer Perforator Triangle Bottle Pipette Beaker

Move

I. Materials and equipment

Leaves of castor, tobacco, sunflower, rape, wheat or cotton.

Model 721 spectrophotometer, vacuum pump (or syringe), incubator, balance, vacuum desiccator, punch, triangular flask, pipette, beaker

Pharmaceuticals

0.1 M phosphate buffer (pH 7.5)

0.2 M KNO3 solution

Sulfanilamide (p-aminobenzenesulfonic acid amine) reagent: 1 g of sulfanilamide is dissolved in 25 ml of concentrated hydrochloric acid and then diluted to 100 ml with distilled water.

α-Naphthylamine reagent: 0.2 g of α-naphthylamine was dissolved in 25 ml of concentrated hydrochloric acid and diluted to 100 ml with distilled water.

NaNO2 standard solution: 1 g NaNO2 dissolved in 1000 ml distilled water to form NaNO2 mother liquor. During the experiment, dilute it to the required concentration according to the treatment. For example, 1 μg/ml: take 1 ml of mother liquor and dilute it to 1000 ml with distilled water.

Experimental steps

1. Wash the freshly retrieved material leaves with water and dry them with absorbent paper. Use a perforator to remove the diameter of 1 cm of the garden piece, and then wash with distilled water 2-3 times, the distilled water will be sucked dry. Weigh two equal portions of leaf garden slices on a balance: about 0.4 g each (or 50 garden slices per portion). Place the two portions of slices in each of the following two solutions, using a 50 ml triangular flask as the container.

(1) 0.1 M phosphate buffer 5 ml + distilled water 5 ml .

(2) 0.1 M phosphate buffer 5 ml + 0.2 M KNO3 solution 5 ml .

Place the triangular flask in a vacuum desiccator and connect a vacuum pump to pump the air so that the garden slice sinks to the bottom of the flask (the garden slice can also be placed in a syringe to pump the air). The triangular flask was placed in a 30°C incubator (in the dark) and kept warm for 30 minutes, then 1 ml of each of the two solutions was taken and prepared for the determination of NO2-content.

(Note that the leaves should have undergone a period of photosynthesis before taking the leaves. The activity of this enzyme is reduced if the carbohydrate content of the tissue is low. When the sugar content in the tissue is low, 30 μ M 3-phosphoglyceraldehyde or 1,6-diphosphofructose can be added to the reaction solution, which can significantly increase the production of NO2-).

2. Plotting the standard curve: The sulfonamide colorimetric method can measure NaNO2 levels below 1 μg/ ml. A standard curve is drawn over the concentration range 0-5 µg/ml. The speed of the color development reaction is related to the speed of diazotization and coupling reaction, so it is affected by the temperature, acidity and alkalinity, etc., which also causes the change of sensitivity. If the standard sample is measured under the same conditions as the measured sample, the speed of color development is also the same, so it can be compared.

Pipet 1 ml of NaNO2 solution of different concentrations in a test tube, (set NaNO2 solution treatment can be 5, 4, 3, 2, 1, 0 micrograms / ml) add 2 ml of sulfanilamide reagent and 2 ml of α-naphthylamine reagent, mix and shake well. Immediately after 30 minutes of holding time in a 30°C incubator, the color was compared on a colorimeter, and the optical density (or transmittance) was read with a No. 50 filter or at a wavelength of 520 nm. The optical density-concentration scale curve was plotted with NaNO2 concentration as the horizontal coordinate and optical density as the vertical coordinate.

3. Determination of NO2-content: place 1 ml of the sample in a test tube, add 2 ml each of sulfanilamide reagent and α-naphthylamine reagent, mix and shake well, and then determine the colorimetry immediately after 30 minutes of rest (in a 30℃ temperature box): read the optical density at 520 mM. The NO2- content was checked by comparing the standard curve. The enzyme activity was then calculated: expressed as micromolar (μM ) values of NO2- produced per gram of fresh weight per hour.

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