Isotopic Fractionation During Reduction of Nitrate and Nitrite by Extracts of Spinach Leaves

Abstract

The isotopic fractionations of nitrogen during the reduction of NO₃^- and NO₂^- in a cytosolic fraction and in a chloroplast preparation from spinach (Spinacia oleracea L.) leaves were determined. The reduction of NO₃^- to NH₃ was studied using a reconstituted system containing cytosolic extract and intact chloroplasts, while a chloroplast system was used for NO₂^- reduction. In the reconstituted systems the ratio of nitrate reductase activity to nitrite reductase activity had a large effect on the relative amounts of NO₂^- and NH₃ formed. Ammonia predominated when the nitrate reductase to nitrite reductase activity ratio was 1 : 5 and this ratio was used in the isotopic fractionation studies. Significant isotopic fractionation of N was observed in the reconstituted system but not in the chloroplast system. This indicates that the observed isotopic fractionation was associated with the reduction of NO₃^- to NO₂^- by nitrate reductase. The isotopic fractionation (i.e. δ¹⁵N_product - δ¹⁵N_substrate) for this reaction was - 15‰.