Method of Measurement of Nitrate Reductase Activity in Field Conditions

For the last three decades the interest in biomonitoring and ecological studies has been rapidly growing. Therefore, it was necessary develop of new methods of analysis for biochemical parameters which allow to quantify biological response of investigated organisms for environmental factors. The main goal of this paper demonstrates optimal conditions for enzyme kinetics analysis conducted in the field in situ. Nitrate reductase activity is typically assayed in vivo by measuring nitrite production in tissue which has been vacuum infiltrated with buffered nitrate solution. For this study a nitrate reductase assay was adapted from a number of studies with own modifications of authors. Leaves of examined plants were collected from the investigated plots and immediately placed into test tubes with buffer solution (potassium phosphate dibasic containing 0.6% propanol-1) and evacuated in 0.33 atm. for 10 minutes. Then, known amount of potassium nitrate was added, and the solution sample was analyzed in order to obtain a background level of nitrite. The foliage samples were incubated for 2 hours at 20 °C in darkness. Following this procedure, they were given the most optimal conditions for reaction stability. After incubation the amount of synthesized nitrite was determined colorimetrically using sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride, measured at 540 nm. The foliage samples were oven-dried to obtain dry mass. The level of nitrate reductase activity was calculated as the amount of nitrite produced in nmol per gram of dry mass of foliage tissue per hour. The result obtained during the research demonstrate the changes of nitrate reductase dynamics according to change of incubation parameters. Dynamics of enzyme activity with changes of solution pH and incubation temperature was presented. Installation for conducting infiltration process and construction of incubation chamber is also described in this paper.