Nitrate-NR

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

1. 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. 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.

3. 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.

4. 1% sulfanilamide: Weigh out 0.15g of sulfanilamide into a small amber bottle. Add 15mL of 3M HCl.

5. 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.