Nitrate reductase activity assay (in vivo method)

Summary

Nitrate reductase activity assays can be used to: measure its efficiency in catalyzing the reduction of nitrate to nitrite in plants.

Operation method

Sulfanilamide colorimetric method

Principle

Nitrate reductase (NR) is a key enzyme in plant nitrogen assimilation, which catalyzes the reduction of nitrate to nitrite in plants, and the resulting nitrite is quantitatively combined with p-aminobenzenesulfonate (or p-aminobenzenesulfonamide) and α-naphthylamine (or naphthylethylenediamine) to produce a red dipole nitrogen compound under acidic conditions. The red azo compounds generated are quantified under acidic conditions. The red azo compounds have a maximum absorption peak at 540 nm and can be measured spectrophotometrically. Nitrate reductase activity can be expressed by the amount of nitrite nitrogen produced. It is generally expressed in units of Nμg-g-1-h-1.The determination of NR can be categorized into in vivo and ex vivo methods. The in vivo method has simple steps and is suitable for rapid, multi-group determinations. The in vivo method is complex, but has better reproducibility.

Materials and Instruments

Crop leaves
Sodium nitrite standard solution Phosphate buffer P-aminobenzenesulfonic acid solution α-naphthylamine solution Trichloroacetic acid solution Isopropyl alcohol Phosphate buffer KNO3
Spectrophotometer Vacuum pump Balance One-sided blade Thermo box Graduated test tube Pipette

Move

1. Standard curve production

Take 7 clean and dry 15ml graduated test tubes and add reagents according to the order of Table 13-1, that is, a series of standard nitrite nitrogen solution of 0-2.0μg is prepared. Shake well in a 30℃ incubator or thermostatic water bath for 30min, and then compare the color at 540nm. Take nitrite nitrogen (μg) as the horizontal coordinate and the optical density value as the vertical coordinate to plot the standard curve or establish the regression equation.

Table 13-1 The order of addition of each reagent

2. Enzyme reaction and enzyme activity determination

(1) Sampling: Wash the material (leaves of wheat, corn and other crops), rinse it with distilled water, and absorb it on filter paper. In the middle of the leaf blade to hit the diameter of 1cm disc (or cut into small pieces of 0.5 - 1. ^0cm2 ), after mixing each sample weighed 0.5 - 1.0g 3 copies, into the test tube and numbered.

(2) Reaction: Add 9 ml of _{KNO3-isopropanol-phosphate} buffer mixture to each tube, and immediately add 1.0 ml of trichloroacetic acid to one of the tubes as a control. Then all the test tubes were put in a vacuum dryer and connected to a vacuum pump for several times until the leaves sank to the bottom of the tubes. The tubes were kept at 30℃ in the dark for 30min, and 1.0ml of TCA was added to the treated tubes, and the enzyme activity was terminated by shaking well.

(3) Colorimetry: The tubes were allowed to stand for 2 min, 2 ml of supernatant was aspirated and added to another set of tubes, the control tube was used as a reference, and the enzyme activity (N ^μg-g-1-h-1 ) was calculated by colorimetric assay according to the standard curve practice.

Where C: total amount of nitrite nitrogen catalyzed by the reaction solution (μg);

V1: volume of buffer added during extraction of enzyme solution (ml);

V2: volume of crude enzyme solution added during enzyme reaction (ml);

W: weight of the sample (g);

t: reaction time (h).

Caveat

Sulfanilamide colorimetric method is relatively sensitive, the speed of color development is susceptible to temperature, acidity and other factors, so it is necessary to strictly control the experimental conditions, the standard solution and the sample reaction solution at the same time reaction, colorimetry.

Common Problems

Nitrate reductase activity can be measured by in vitro and in vivo methods, which are also known as in vitro and in vivo methods, respectively. The in vivo method is simple and rapid, and has been adopted more and more. The in vivo method has the disadvantages of low NRA values and unsatisfactory reproducibility, so it is necessary to further explore the in vivo method to determine the optimal measurement conditions for different materials.

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