Otimização do método para a análise da enzima redutase do nitrato na cultura da soja

Abstract

Enzyme analysis is a valuable tool and is currently in increasing demand to identify nutritional imbalances in crops in the field and in technical-scientific research. Each plant species has its anatomical and physiological particularities, which makes it necessary to establish appropriate analytical conditions for each. Thus, the establishment of appropriate analytica condition for soybean (Glycine max) crops will contribute to improving the operability and quality of analyzes at the CPMUA (Centro de Pesquisa Multiuser do Araguaia). Thus, the objective of this work was to standardize the optimal conditions for determining the activity of the enzyme nitrate reductase in fresh soybean leaves. Four samples of pre-flowering diagnostic leaves were evaluated at random points within the cultivation area, where the analyzes of each test took place (one test per day, in a total of five). The activity of the enzyme nitrate reductase was determined using an in vivo method, where 300 mg of leaf tissue fragments were placed in test tubes with 4 mL of potassium nitrate (0.25 mol L-1), buffer of phosphate (pH 7.4), composed of monosodium phosphate (0.20 mol L-1) and dibasic sodium phosphate (0.50 mol L-1). At the end of the incubation time, an aliquot of 1 mL of the plant extract was removed and the aliquot mixed with adequate amounts of 1% sulfanilic acid in 2N hydrochloric acid, alpha naphthylamine (193 mmol L-1) and sodium acetate, for later determination of the absorbance in a spectrophotometer. The results obtained were submitted to analysis of variance (F test). The tests with conditions that characterize qualitative variables were compared by means test and the quantitative ones by regression, with the aid of the Sisvar program. The conditions for carrying out the different experiments for the optimization of the method for determining the activity of the enzyme nitrate reductase in soybean should be composed of: cut type (cut disc); incubation time of 2 h; 0.4 g of leaf tissue; concentration of 0.25 M KNO₃ and temperature of 35 °C in the dark.