Nitrate reductase method¶
Reagents & Equipment¶
- Colorimeter
- Cuvettes
- 5 mL sample bottles or other capped tubes
- Potassium phosphate (KH2PO4)
- Potassium hydroxide (KOH)
- 25 mM EDTA
- 2 units Nitrate reductase (NR), NECi Cat #
- NADH
- Sulfanilamide, Sigma Part #
- NED
- (Optional) Distilled water for sample dilutions
Colorimeter set-up¶
- Wavelength: 528 nm. Green led (colorimeter RGB board)
- Nitrate standard data: download a data file or prepare your own nitrate standard data
- Before measuring blank the colorimeter against water or a distilled water sample developed with API reagent as described below
Method¶
Step 1: Prepare reagents¶
- Phosphate buffer, pH7.5 (Phosphate buffer (25 mM KH2PO4, 0.025 mM EDTA): Dissolve 3.75 g of potassium phosphate (KH2PO4) and 1.4 g potassium hydroxide (KOH) in 800 mL of distilled water in a 1L volumetric flask. Add 1 mL of 25 mM EDTA and fill to the 1L mark with distilled water.
- 2 units/mL Nitrate reductase: Add 1 mL of NECi proprietary enzyme diluent to 2 units of freeze-dried enzyme following the instructions with the enzyme.
- 1 mg/ml NADH: Dissolve 0.1 g of NADH (FW=709.4) in 100 mL distilled water. Aliquot and store unused NADH in the freezer.
- 1% sulfanilamide: Weigh out 0.15g of sulfanilamide into a small amber bottle. Add 15mL of 3M HCl.
- 0.02% NED: Weigh out 2 mg (0.02 g) of NED into an amber bottle. Add 100 mL of distilled water.
Step 2: Prepare enzyme assay mix.¶
Prepare a “master mix” of enzyme, NADH and phosphate buffer. Make sure you have enough for all of your samples. The Table below can be used for a 10X master mix for 9 samples.
| Reagent | 1X Mix | 10X Mix |
|---|---|---|
| Phosphate buffer | 890 uL | 8.9 mL |
| 1 mg/mL NADH | 100 uL | 1.0 mL |
| 2 Units/mL NR | 10 uL | 0.1 mL |
| Total volume | 1000 uL | 10.0 mL |
Step 3: Assay method¶
- Transfer 1 mL of the master mix to clean sample tube e.g. glass test tube.
- Add 50 µL of water sample and mix thoroughly
- Incubate at RT for 20-30 minutes.
- Add 500 µL of 1% sulfanimide and mix
- Add 500 µL of 0.02% NED and mix
- Place the sample into a cuvette in the colorimeter and click measure. The program will return the Absorbance and nitrate concentration.