Soil Nitrite Reductase Activity Assay Kit - Spectrophotometric Method

Product Information

Product code: 67051

Soil nitrite reductase is a key enzyme in denitrification. It participates in the reduction of nitrite to NO, and its activity reflects nitrogen transformation efficiency during biodegradation. This assay provides a basis for studying nitrogen transformation patterns.

Nitrite reductase reduces NO₂^-to NO, decreasing the amount of NO₂^-in the sample that participates in the diazotization reaction to form a purplish-red compound. Therefore, the change in absorbance at 540 nm reflects soil nitrite reductase activity.

Package Contents and Storage

Quality and Safety Information

Transportation and Storage

Transportation: This product is transported with ice packs.

Storage: Store according to the instructions in this manual. Shelf life: 180 days.

Instructions for Use

1. Sample Processing

Air-dry fresh soil samples naturally or in a 37°C oven, then pass through a 30-50 mesh sieve.

2. Reagent Preparation

• Reagent 2: Before use, add 12 mL distilled water to dissolve. Store at 4°C for up to 2 weeks or at -20°C for up to one year. After use, promptly store at 4°C or -20°C.
• Reagent 3: If precipitation occurs, heat at 60-80°C to dissolve.
• Reagent 4: If precipitation occurs, heat at 60-80°C to dissolve.
• Standard: 10 μmol/mL sodium nitrite.
• Working solution: Before use, mix Reagent 4 and Reagent 5 at a 1:1 ratio according to the required amount. Prepare fresh before use. After preparation, the working solution is valid for 48 h at 4°C.

3. Operating Procedure

1. Preheat the spectrophotometer for 30 min or longer. Set the wavelength to 540 nm and zero with distilled water.
2. Dilute the 10 μmol/mL standard solution with distilled water to prepare 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0078125, and 0.001953125 μmol/mL standard solutions for testing.

Standard Solution Dilution

In the following experiment, each standard tube requires 350 μL standard solution. Do not directly measure absorbance at this dilution step.

4. Measurement Procedure

Mix thoroughly. Pipette 1 mL reaction solution into a 1 mL glass cuvette and measure absorbance at 540 nm. Record the readings as A_{no soil}, A_{blank 1}, A_control, A_measurement, A_standard, and A_{blank 2}.

Calculate: ΔA_measurement= (A_soil-free- A_{blank 1}) - (A_measurement- A_control)

Calculate: ΔA_standard= A_standard- A_{blank 2}

The soil-free tube, blank tube 1, and standard curve only need to be performed 1-2 times.

5. Calculation

5.1 Standard Curve

Use the standard tube concentration (X, μmol/mL) and absorbance (Y, ΔA_standard) to establish the standard curve. Substitute ΔA (Y, ΔA_measurement) into the standard curve formula to calculate the sample concentration (X, μmol/mL).

5.2 S-NiR Activity

Definition of enzyme activity unit: the amount of enzyme required for each g soil sample to reduce 1 μmol NO₂^-per day is one enzyme activity unit.

S-NiR (U/g) = X × V_reaction÷ W ÷ T = 14.4 × X ÷ W

• V_reaction: total volume of enzymatic reaction, 0.6 mL
• T: reaction time, 1 h = 1/24 d
• W: sample mass, g

Precautions

1. Before the formal assay, select 2-3 samples with large expected differences for a pre-experiment. This 50T kit can test 24 samples.
2. Required instruments and supplies to be prepared by the user: balance, visible spectrophotometer, 1 mL glass cuvette, adjustable pipette, benchtop centrifuge, oven, 30-50 mesh sieve, constant-temperature water bath or incubator, and distilled water.
3. If ΔA_measurement> 2.5, dilute the sample before measurement and modify the calculation formula accordingly.
4. The linear detection range of this kit is 0.001953125-0.5 μmol/mL.

Appendix

The standard curve should be prepared by the customer for greater accuracy. Refer to the operating table above for the steps. The customer may use the standard curve formula or use the absorbance values of each standard well obtained according to the operating table to prepare a standard curve with R²≥ 0.99, then obtain the calculation formula for sample calculation.