A Major Error in the Acetylene Reduction Assay: Decreases in Nodular Nitrogenase Activity Under Assay Conditions

Observations on both attached nodulated roots and detached noduleshave revealed that nitrogenase activity in many legume speciesdeclined rapidly in the presence of acetylene, with a concurrentreduction in respiration. The reduction began within a few minutesof exposure to acetylene and continued for 30–60 min beforea new steady-state was attained. A similar decline in H₂ evolutionand respiration was observed when N₂ was replaced with argonor helium. This suggests that the decrease is linked to thecessation of ammonia production. Measurements of ¹⁵N₂ uptakedemonstrated that it is the pre-decline rather than final rateof ethylene production which represents the real rate of nitrogenaseactivity. The implications of these findings for the interpretationof acetylene reduction and hydrogen evolution data are considered. Key words: Roots, Acetylene, Nitrogenase activity