Nitrate Reductase Activity

Nitrate reductase was assayed using the method described by the Nitrate Elimination, Co., Inc. (NECi³) with some modifications. Crude protein was extracted from the ground tissue using an extraction buffer containing 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS) pH 7.5, 1.0 mM EDTA and 10 mM L-cysteine. PVPP [1% (w/v)] was added to the grinding mixture during extraction. Four mL of extraction buffer was used per gram fresh weight plant tissue. The homogenate was passed through four layers of cheesecloth and centrifuged at 21000 g for 5 min at 4°C. The supernatant was used in subsequent assays. Approximately 100–200 μg of the extracted protein was added to 800 μL of substrate solution (30 mM potassium nitrate in 100 mM MOPS, pH 7.5). The reaction was started by adding 100 μL of 25 mM NADH and stopped after 10 min by adding 100 μL of 100 mM zinc acetate. After centrifuging at 22000 g for 2 min, 500 μL of the supernatant was add to a fresh 1.5 mL tube. To this, 500 μL (an equal volume to the volume of supernatant) was added of each of the color development reagents [1% (w/v) sulfanilamide in 1.5 N HCl and 0.02% N-(napththyl)-ethylenediaminehydrochloride]. Samples were left at room temperature for 10–20 min to allow full color development. Absorbance was read at 540 nm. Nitrite concentration was estimated using a standard curve prepared by diluting known concentrations of nitrite in 500 μL and adding the color development reagents.