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Improvements of the Nitrite Color Development in Assays of Nitrate Reductase by Phenazine Methosulfate and Zinc Acetate ¹

^a Department of Agronomy, University of Illinois, and United States Regional Soybean Laboratory, Urbana, Illinois 61801

Nitrate reductase activity is most commonly assayed by measurement of product formation. Excess NADH and factor(s) present in the enzyme extract that interfere with the diazotization and azo color complex of nitrite cause a depression of apparent nitrate reductase activity. Two postassay treatments were found that markedly enhanced the extent of nitrite color formation and apparent nitrate reductase activity. The procedure involves stopping the reaction with zinc acetate (50 µmoles per ml of reaction mix), followed by removal of the precipitate by centrifugation. Presumably the zinc acetate removes extract factor(s) that interfere with color development, because it does not remove the NADH. Phenazine methosulfate (15 nmoles per ml of reaction mix) is added to aliquots of the supernatant and allowed to stand for 20 min at 30 C to oxidize the residual NADH before color development.

¹ Cooperative investigation of the Illinois Agricultural Experiment Station and North Central Region, Agricultural Research Service, United States Department of Agriculture. Research was supported in part by Rockefeller Foundation Grant AGR 7114 and a United States Department of Agriculture Cooperative Agreement 016-15-18.

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