Nitrate Reductase Assay

Method: The method employed is that of Lowe and Evans (1964) wherein the reaction velocity is determined by measuring the production of nitrite in a dithionite/methyl viologen system. One unit produces one micromole of nitrite per minute at 30°C and pH 7.0 under the specified conditions.

Note: Enzyme and test are destroyed by oxygen. Extreme care must be taken when mixing and adding reagents. The test should be dark blue to show that it is in a reduced conditon until it is stopped by shaking.

Prepare: Ice bath (used to slow oxygen gain)
30°C dry bath with thermometer
12 X 75 test tubes
Serological pipets, 1 and 2 ml
Vacuum with 4 ports
4 side arm vacuum flasks with stoppers

Reagents

Reagent grade water: Place 150 ml water in 250 ml side arm and stopper. FLASK #1.
0.10 M Sodium nitrate: Dissolve 850 mg of sodium nitrate (MW 84.99) in 100 ml reagent grade water. Put in side arm flask and stopper. FLASK #2.
0.15 M Potassium phosphate, pH 7.0: Dissolve 2.04 gm potassium phosphate, monobasic (MW 136.1) in 80 ml reagent grade water. pH to 7.0 with NaOH, q.s. to 100 ml. Put 80 ml in side arm flask and stopper. FLASK #3. Save remaining 20 ml.
20 ml Phosphate buffer/40 ml water: Place 40 ml reagent grade water and the remaining 20 ml of 0.15 M potassium phosphate, pH 7.0 in side arm flask and stopper. FLASK #4.
58 mM Sulfanilamide in 3N HCl: Dissolve 199.8 mg of sulfanilamide (MW 172.21) in 20 ml of 3N HCl.
0.10 M Sodium nitrate
23 mM Sodium dithionite (MW 174.11) prepared in 48 mM sodium bicarbonate. Prepare fresh and avoid aeration.
0.39 mM N-(1-napthyl) ethylenediamine hydrochloride (NED): Dissolve 10.1 mg of NED (MW 259.12) in 100 ml reagent grade water.
1000 umole/L Sodium nitrite stock solution: Dissolve 69 mg of sodium nitrate (MW 69.0) in 1 L of reagent grade water.
Dry reagents: Weigh 80 mg Sodium dithionate (MW 174.11), 80 mg sodium bicarbonate (MW 84.01), and 5 mg methyl viologen into a small test tube.

Procedure

Connect all four filled vacuum flasks to vacuum, place in ice bath and de-gas for 30 minutes. Check 30°C bath. Number tubes and set up in ice bath. Have one tube with 0.5 ml H₂O and thermometer as a temperature check. After 30 minutes under vacuum, release vacuum. Add weighed dry reagents (dithionite, bicarbonate, methyl viologen) to degassed flask containing H₂O/PO₄^-3. (FLASK #4) Replace stopper. Swirl gently to dissolve. Should be deep blue and stay that way. Do not put back under vacuum once dry reagents have been added. If color fades discard. Prepare enzyme and then continue procedure.

Enzyme

Prepare enzyme at 10 mg/ml in degassed water just prior to use. Use serological pipets to gently add water, cover, and mix gently. Further dilutions are made in degassed KPO₄ buffer. Enzyme is not stable. Run 2X and 4X dilutions.

Procedure, continued

Set up 9 tubes, labelled as listed in next column. Pipet water, nitrate, blue reagent, nitrite (std) into respective tubes using serological pipets.

Tube	Water degassed ml	Nitrate degassed ml	Blue reagent degassed ml	Nitrite umoles
1 - blank	0.01	-----	0.30	-----
2 - std (0.02)	0.08	-----	0.30	0.02
3 - std (0.05)	0.05	-----	0.30	0.05
4 - std (0.10)	-----	-----	0.30	0.10
5 - substrate blk	-----	0.10	0.30	-----
6 - test 1 2X	-----	0.10	0.30	-----
7 - test 1 4X	-----	0.10	0.30	-----
8 - enz blk 2X	0.10	-----	0.30	-----
9 - enz blk 4X	0.10	-----	0.30	-----

Transfer to 30°C bath and watch temperature. As soon as temperature reaches 30°C, at timed intervals add 0.1 ml diluted enzyme to tubes 6-9. Incubate for 10 minutes. Reaction mixture should still be deep blue.

After 10 minutes, stop reaction by vigorous mixing until blue color is completely removed. Quickly add 0.5 ml of sulfanilamide solution and 0.5 ml of NED solution. Add 1.5 ml reagent grade water and incubate at room temperature for 10 minutes. Read A₅₄₀ nm.

Calculation

From the standards calculate ΔA₅₄₀ /umole. (i.e. 0.02 umole = 540nm of 0.306 = 0.306/.02 = 15.3). Take average of three points.

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