Manual Nitrate Reductase Nitrate Analysis
Manual nitrate analysis is carried out by
reconstituting freeze-dried AtNaR2 (EC 1.7.1.1) in
25 mM potassium phosphate, 0.1 mM Na2EDTA, pH 7.5, at 22-25°C, to a final
concentration of 1 unit per mL. A total of 3 units of NaR are needed to
analyze approximately 50 water samples and nitrate standards.
The unit of enzyme activity is defined as the
amount of NaR catalyzing conversion of 1 µmol nitrate to nitrite per min
at pH 7.5 and 30°C.
The Nitrate Analysis Reagent is prepared by
bringing the reconstituted AtNaR2 solution (3 units) to 54 mL by mixing
with additional phosphate buffer, and the addition of 1.0 mL of 2.82 mM
NADH solution, prepared in deionized water. Final volume of the Nitrate
Analysis Reagent = 55 mL. This reagent is stable for about 1 hour and
should be used immediately after the NADH is added.
The enzymatic reduction step in the nitrate
analysis method is carried out by adding 50 µL of aqueous sample or
nitrate standard to 1.0 mL of nitrate analysis reagent, mixing completely
with a test tube mixer, and incubating the mixture at 37°C for 15 min in a
water bath. Reagent blanks are also prepared by replacing the water sample
in the above step with 50 µL of deionized water. The length of the
incubation does not need to be timed exactly and no harm is done by
leaving the mixture reacting for up to 2 hours.
The color development step is carried out by
addition of 100 µL of sulfanilamide reagent (10 g per L of ~1.2 N HCl),
with vigorous mixing and a 2 min reaction time, which is followed by
addition of 100 µL N-(1-naphthyl)ethylenediamine dihydrochloride reagent
(1.0 g per L of deionized water) and reacting mixture at 22-25°C or 37°C,
for at least 5 min. A longer reaction time is not harmful as long as the
absorbance of the samples are read within 1 hour.
Finally, the absorbance at 540 nm was
determined for each sample and nitrate standard using a Hewlett-Packard
spectrophotometer (or any colorimeter or spectrophotometer capable of
reading absorbance at 540±20 nm is OK.
A standard curve is generated from the
absorbance of nitrate standards prepared in the range of 0.2 to 10 ppm
nitrate-N using a certified 1000 ppm nitrate standard (Fisher Scientific
Co.). See example of the standard curve in the Results section of
NaR-Nitrate Analysis Method.
The nitrate content of the samples is
determined from their absorbance using the nitrate standard curve, which
is easily done using EXCEL® or another spreadsheet program. See
example of Nitrate Content in the Results section of
NaR-Nitrate Analysis Method.
Nitrate-free deionized water must be
used, and all containers and reaction vessels must also be free of
nitrate. If you have a high background color in all samples, even
your control reagent blanks |
