Nitrate reductase activity test: phenazine methosulfate-ferricyanide stop reagent replaces postassay treatment |
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| Authors: | L Scheideler H Ninnemann |
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| Institution: | 1. Departamento de Química, Universidade Federal de Lavras, Campus Universitário, CP 3037, 37200-000 Lavras, MG, Brazil;2. Departamento de Química, Universidade Federal de Minas Gerais, Campus Pampulha, 31.270-901 Belo Horizonte, MG, Brazil;1. Department of Chemistry, Hakim Sabzevari University, Sabzevar, 96179-76487, Iran;2. Department of Chemistry, Tehran North Branch, Islamic Azad University, Tehran, Iran;3. Department of Chemistry, Payame Noor University (PNU), Tehran, 19395-4697, Iran;4. College of Chemistry, Shahrood University, Shahrood, 36155-316, Iran |
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| Abstract: | Nitrate reductase activity is usually measured by colorimetric determination of the nitrite formed. Since reduced pyridine nucleotides interfere with color formation, the use of NADPH or NADH in the assay requires a specific postassay treatment to remove excess substrate. A "stop mix" containing 1.5 mM phenazine methosulfate and 4.0 mM ferricyanide (final concentrations 0.136 and 0.36 mM, respectively) can remove excess NAD(P)H and terminate the enzymatic reaction quickly in a single, time-saving step. For activity tests containing dithionite we recommend the use of a 1:1 mixture of the two color reagents to avoid incomplete color formation. This may occur during longer time intervals between addition of the color reagents due to destruction of the diazonium salt formed with the first reagent by oxidation product(s) of dithionite. |
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