(NADH: Nitrate Oxidoreductase EC 22.214.171.124 (NADH-dependent))
(NAD(P)H: Nitrate Oxidoreductase EC 126.96.36.199 (NAD(P)H-dependent))
(NADPH: Nitrate Oxidoreductase EC 188.8.131.52 (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.
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.
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.
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