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Stable  15 N isotopes have been used to examine movement of nitrogen (N) through various pools of the global N cycle. A central reaction in the cycle involves the reduction of nitrate (NO −  3 ) to nitrite (NO −  2 ) catalyzed by nitrate reductase (NR). Discrimination against  15 N by NR is a major determinant of isotopic differences among N pools. Here, we measured  in vitro   15 N discrimination by several NRs purified from plants, fungi, and a bacterium to determine the intrinsic  15 N discrimination by the enzyme and to evaluate the validity of measurements made using  15 N-enriched NO −  3 . Observed NR isotope discrimination ranged from 22 to 32‰ (kinetic isotope effects of 1.022–1.032) among the different isozymes at natural abundance  15 N (0.37%). As the fractional  15 N content of substrate NO −  3  increased from natural abundance, the product  15 N fraction deviated significantly from that expected based on substrate enrichment and  15 N discrimination measured at natural abundance. Additionally, isotopic discrimination by denitrifying bacteria used to reduce NO −  3  and NO −  2  in some protocols became a greater source of error as  15 N enrichment increased. We briefly discuss potential causes of the experimental artifacts with enriched  15 N and recommend against the use of highly enriched  15 N tracers to study N discrimination in plants or soils.

Nitrate reductase 15N discrimination in Arabidopsis thaliana, Zea mays, Aspergillus niger, Pichea angusta, and Escherichia coli