Methodology for Enzymes

Nitrate Reductase
(NADH: Nitrate Oxidoreductase EC (NADH-dependent))
(NAD(P)H: Nitrate Oxidoreductase EC (NAD(P)H-dependent))
(NADPH: Nitrate Oxidoreductase EC (NADPH-dependent))

The assimilatory reduction of nitrate by plants is a fundamental biological process in which a highly oxidized form of inorganic nitrogen is reduced to nitrite and then to ammonia.
NO-3 + AH2

NO-2 + A + H2O

The nitrate reducing system consists of nitrate reductase and nitrite reductase which catalyze stepwise reduction of nitrate to nitrite and then to ammonia. According to the specificity of electron donor two major types of nitrate reductase occur:
a) Ferredoxin-dependent nitrate reductase (blue-green algae)
b) Pyridine nucleotide-dependent nitrate reductase (higher plants)

Nitrate reductase (NR) is capable of utilizing the reduced form of pyridine nucleotides, flavins or benzyl viologen as electron donors for reduction of nitrate to nitrite. NADH-dependent nitrate reductase is most prevalent in plants. Hence, NR activity in plants can be measured by following the oxidation of NAD(P)H at 340nm. However, NR   activity is commonly measured by colorimetric determination of nitrite produced.

» Potassium Phosphate Buffer 0.1M (pH 7.5)
» Potassium Nitrate 0.1M. Dissolve 1.01g potassium nitrate in 100mL water.
» NADH 2mM. Dissolve 14mg NADH disodium salt in 10mL water.
»  Sulphanilamide, 1% (w/v). Dissolve 1g sulphanilamide in 100ml 2.4N hydrochloric acid.
» N-(1-naphthyl) Ethylenediamine Dihydrochloride, 0.02%. Dissolve 20mg in 100mL water.
» Potassium Nitrite Standard Solution (0.01M).
   Dissolve 851mg pure potassium nitrite in 100mL water in a standard flask.
   Dilute 10mL of this solution to 100mL and use as working standard solution.
» Enzyme Extract
   Homogenize a weighed quantity of the plant material in a known volume of medium (6mL for 1g fresh tissue) containing 1mM EDTA, 1-25mM cysteine and 25mM potassium phosphate adjusted to a final pH 8.8 with KOH. Filter through four layers of cheese cloth and centrifuge for 15 min at 30,000g. Decant the supernatant through glass wool and use for assays. Extract under ice-cold conditions.

Pipette out 0.5mL phosphate buffer (pH7.5) in a test tube.
Add 0.2mL potassium nitrate solution, 0.4mL NADH solution and 0.7mL water.
Initiate the reaction by the addition of 0.2mL enzyme extract. Set up a control in the same way but with water instead of enzyme extract.
Incubate at 30°C for 15 min.
Terminate the reaction by the rapid addition of 1mL sulphanilamide followed by 1mL naphthyl ethylenediamine reagent.
Wait for 30 min.
Measure the absorbance at 540nm.
Prepare a standard graph with sodium nitrite. Pipette out different known aliquots of potassium nitrite standard solution into a series of test tubes and make up the volume in each tube to 2mL by adding water. Proceed from step 5 to 7.

Activity is expressed as micromole nitrite produced per min per mg protein (or per g fresh tissue).

1.  The enzyme reaction rate is linear over a 30 min period.
2.  The pink color produced by nitrite is stable for 2-3h.

1.  Hageman, R H and Reed, A J (1980) In: Methods in Enzymology. Vol. 69 Part C (Ed Anthony San Pietro) Academic Press New York p 270.
2.  Sadasivam, S and Gowri, G (1981) Experientia 37 552.
3.  Snell, F D and Snell, C T (1949) Colorimetric Methods of Analysis 2 Van Nostrand Co Inc New Jersey p 805.