10 Assay of nitrate reductase (NR) enzyme  
 

 

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Nitrate Reductase (EC1.6.6.1) is a cytoplasmic metalloenzyme primarily expressed in root epidermal and cortical cells and shoot mesophyll cells. NR catalyses the transfer of two electrons from NAD(P)H to nitrate to produce nitrite.

 

Materials and Reagents

 

Potassium phosphate (monobasic)

EDTA sodium salt

Potassium nitrate

Potassium nitrite

NADH

Sulphanilamide

N-(1-naphthyl ethylene diamine dihydrochloride (NEDDH)

Concentrated HCl

Spectrophotometer

pH meter

Quartz cuvettes

 

Composition of reaction mixtures
 

A. 100 mM Potassium phosphate buffer pH 7.3 with EDTA (Extraction buffer)

Reagent A: Dissolve 387 mg of EDTA sodium salt in 10 ml of dH2O

Reagent B: Dissolve 1.361 g K2HPO4 (monobasic) in 50ml of double distilled water

Add 200 Ál of Reagent A to 50 ml of Reagent B

Make the volume upto 100 ml with dH2O

Pre-chill before use.

B. 30mM Potassium nitrate

Dissolve 303.33 mg of KNO3 in 100ml dH2O

Pre-chill before use

C. 2.5 mM NADH

Dissolve 100 mg of NADH in 56.4 ml of dH2O

Store under refrigeration.

D. 58 mM sulphanilamide

Dissolve 1g sulphanilamide in 24.7 ml of 35.4% con. HCl (2.4N)

Make volume up to 100 ml with dH2O

E. 0.77 mM NEDDH

Dissolve 20 mg NEDDH in 100 ml dH2

Procedure

 

1.   Freeze dry fresh live plant samples in liquid nitrogen and powder in a mortar using a pestle.

2.   Weigh 250mg of the sample into a 2ml eppendorff tube containing 1ml of pre-chilled extraction buffer.

3.   Mildly shake the extraction mixture and centrifuge at 14000 rpm for 10 minutes.

4.   Pipette out 200Ál of the supernatant crude extract to a sterile clean test tube containing 1.0 ml of 100 mM of extraction buffer

5.  Add 1.6 ml of 30 mM potassium nitrate solution and 200 Ál of 2.5 mM NADH into the test tube. 

6.  Incubate the assay mixture for 30 min under dark with mild shaking.

7.  Immediately after incubation, stop the reaction by adding 1 ml of 58 mM sulphanilamide.

8.  Add 1 ml of 0.77 mM NEDDH in the tubes

9.  The red color developed is read in the spectrophotometer at 540nm.

10. Maintain a blank by using 200 Ál of dH2O instead of the enzyme extract

11. Prepare a standard curve by using potassium nitrite standards of ÁM concentrations of 0, 20, 40, 60, 80 and 100 and using 200 Ál of these solutions instead of the enzyme extract in the above procedure, but without incubation.