Nitrate reductase (NR):

Nitrate reductase (NR; EC 1.6.6.1) activity will be performed by measuring the amount of NO2 formed in NR assay buffer (Wray and Filner, 1970). The assay medium for NR contained 0.5 ml potassium phosphate buffer (100 mM, pH 7.5), 0.1 ml KNO3 (100 mM), 0.1 ml NADH (0.15 mM) and 0.1 ml extract in a final volume of 2.0 ml. The assays will be conducted at 30° C for 30 min. The reaction will be terminated by the addition of 0.1 ml of 1 M zinc acetate, and excess NADH will be oxidized by phenazine methosulfate (Scholl et al., 1974). The NO2 produced will be assayed after diazotization with 1 ml of sulfanilamide (1 g dissolved in 1.5 N HCl) and 1 ml N -naphthyl ethylene-diamine-dichloride (NED, 0.2 g  in double-distilled water). The solution mixture will be centrifuged at 12,000 g for 5 min, and the absorbance of the supernatant will be determined at 540 nm. The enzyme activity will be measured as mmol of nitrite produced. Nitrite reductase (NiR; EC 1.6.6.4) activity will be measured by NO2 consumption during the assay (Losada and Paneque, 1971). The assay mixture contained 0.5 ml potassium phosphate buffer (100 mM, pH 7.5), 0.2 ml potassium nitrite (15 mM), 0.3 ml methyl viologen (5 mM), and 0.1 ml extract in a total volume of 1.1 ml. The reaction will be initiated by adding 0.2 ml sodium dithionite (Na2S2O4) [86.15 mM sodium dithionite dissolved in 290 mM sodium bicarbonate (NaHCO3)]. A blank without sodium dithionite also will be run simultaneously. After incubation for 30 min at 30 C, the reaction will be stopped by vigorously mixing the content of the assay tube on a vortex mixer until the methyl viologen will be oxidized completely (10–15s). The residual NO2 in the reaction mixture will be determined colorimetrically as described above. The enzyme activity will be measured as mmol of nitrite reduced g h-1.

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