Improvements of the Nitrite Color Development in Assays of Nitrate Reductase by Phenazine Methosulfate and Zinc Acetate

Abstract

(14), is laborious and not always reliable, as discussed later. While dialysis removes excess NADH (6), it requires specialized equipment. Enzymatic oxidation is also satisfactory (5), except for time and expense. Phenazine methosulfate rapidly oxidizes NADH (2, 7, 8), but it has not been used to oxidize excess NADH in the NR assay.The objectives of this study were to test the effectiveness of PMS in the oxidation of excess NADH in the NR assay medium before color development and to determine if PMS could be used in conjunction with zinc acetate to improve NO2-recovery in the NR assay.The most sensitive method for assaying nitrate reductase in plant extracts is by colorimetric measurement of the product, nitrite (12). Nitrite is diazotized with sulfanilamide and then reacted with N-(l-naphthyl)ethylenediaminedihydrochloride to produce the azo dye which is measured spectrophotometrically at 540 nm. However, excess NADH from the reaction medium interferes with the full development of color (9) MATERIALS AND METHODS Corn (Zea mays L.) and soybeans (Glycine max L. Merr.) were used as the source of NR for these studies. Leaves from corn seedlings, variety "Oh43 X B14", were harvested after 4 to 5 hr of illumination, and NR was extracted and assayed by methods similar to those described by Hageman and Hucklesby (3). The exceptions were that the fresh leaf tissue was ground in a TenBroeck homogenizer in 10 volumes of extraction medium. Standard (reference) assays, containing 50 ,umoles of potassium phosphate (pH 7.5), 20 ,umoles of KNO,, 0.8 ,umole of NADH, and 0.2 ml of the crude extract in a final volume of 4 ml, were incubated 15 min at 30 C. Blank assays were identical except NADH was omitted. The reactions were terminated by boiling for 2 min, and NO,-color was developed by adding an equal volume of a 1

Nitrate Reductase Activity in Soybeans (Glycine max [L.] Merr.)

“…The lack of a significant response to the addition of NADH may have been due to the large size of the molecule which prevented its transport across cell membranes during or following vacuum infiltration (5, 6). Excess NADH, which interferes with NO2-color development, was removed with PMS as previously reported (9). In contrast to NADH, 100 mm glucose addition to the in vivo NR assay medium increased leaf NR activity 36 and 95% with plants pretreated to 10-hr dark periods at 20 and 30 C, respectively (Table I).…”

“…In vivo NR activity was determined as previously described (8). In vitro NR activity was assayed as described by Scholl et al (9), with the exception that the crude (uncentrifuged) extract was assayed.…”

Soluble and membrane‐associated nitrate reductases in the dinoflagellate Peridinium gatunense

“…After incubation for 1 h at 17mC, the reaction was stopped by boiling for 2 min. When cooled to 17mC, 6 nmol of phenazine methosulphate were added in each tube to remove NADH (Scholl et al, 1974). After 20 min in complete darkness, the reagents for NO # − determination were added in sequence (Snell & Snell, 1949), the tubes were centrifuged (5 min at 10 800 g) and A &%!…”

Nitrate Assimilation in the Basidiomycete Yeast Sporobolomyces roseus

“…Extracts were clarified by centrifugation at 40000g for 20 min at 3 "C. Assays containing extract (about 500 pg protein), potassium phosphate buffer pH 7.5 (50 ymol), K N 0 3 (50 pmol), FAD (2.5 nmol) and NAD(P)H (0.3 mM) were initiated by the addition of NAD(P)H, allowed to proceed for 10 min at 30 "C and terminated by boiling for 2 min. After cooling, residual NAD(P)H was oxidized with phenazine methosulphate (Scholl et al, 1974) and nitrite was estimated.…”