Limitations to the Measurement of In Vitro Nitrate Assimilation by Exogenous Additives and Endogenous Interference Factors in the Leaves of Zea mays L. Seedlings

An evaluation of existing assay procedures for the measurement of nitrate assimilation in the leaves of Zea mays L. has highlighted limitations in established in vitro assay techniques. Both exogenously added compounds and endogenous leaf components affected the results of an in vitro nitrate reductase (NADH: oxido-reductase, EC 1.6.6.1.) assay. Reducing agents employed as enzyme protectants were excluded from the assay in order to accurately measure the concentration of nitrogen compounds by colorimetric and HPLC analysis. Endogenous nitrate levels in a leaf extract as measured by these two analytical techniques indicated significant interference in the colorimetric method due to the presence of various organic compounds. This interference was most apparent at low nitrate concentrations, however, changes in nitrate concentration appeared to be more closely comparable between the two techniques. In addition, endogenous leaf components also interfered with the precise determination of nitrite that had accumulated during an in vitro nitrate reductase assay. These endogenous factors acted directly upon the colorimetric assay of nitrite by a concentration-dependent reaction with the diazotizing reagent sulphanilamide. The interfering components were of low molecular weight ( 5000 daltons) and easily separable from nitrate reductase by molecular sieve chromatography. Their interference in the nitrite assay could only be partially prevented by heating or storage, while other treatments studied, including those frequently used to terminate an in vitro assay such as zinc acetate precipitation or chloroform extraction, had less effect in alleviating the interference. Similar endogenous components which affected the colorimetric assay of nitrite were also found in leaf extracts from wheat, pea, soybean and sunflower seedlings.