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Abstract
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 in cubation chamber is also described in this paper.
Highlights
Nitrogen is known as one of the most abundant biogens on Earth, lack of this nutrient is a widely known phenomenon in many ecosystems
Two factors influencing nitrate reductase activity were examined: pH of incubation buffer and buffer temperature
The obtained results demonstrate no statistical differences of nitrate reductase activity with different buffer reaction F = 0.0011, p = 0.97
Summary
Nitrogen is known as one of the most abundant biogens on Earth, lack of this nutrient is a widely known phenomenon in many ecosystems It is one of the most important factors limiting plant growth [10, 11]. Free nitrogen may be fixed from the atmosphere by some prokaryotic organisms, both free living and symbiotic [9, 15] this form of nitrogen is not available to vascular plant species. It is available in the form of nitrate and/or ammonia, which may be absorbed by tissues from soil or from atmospheric fallout [13, 14]. The presented research tried to find optimal conditions for enzyme kinetics activity with experiment conducted in the field in situ
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