Assays of Nitrogenase Reaction Products

Abstract

Steady-state assays of nitrogenases share at least five requirements: an anaerobic environment, a consistent source of magnesium adenosine triphosphate (MgATP), a suitable source of reductant, a buffer system compatible with the product-quantification protocol to be used, and the desired substrate. The assay is initiated by injection of the component protein(s) of the enzyme or MgATP and terminated by injection of either acid or a solution of Na2EDTA. The various nitrogenases catalyze the reduction of a wide variety of substrates. This chapter outlines the methods used to analyze the products of nitrogenase-catalyzed reactions involving nitrogen–nitrogen bonds, nitrogen–oxygen bonds, carbon–nitrogen bonds, carbon–carbon bonds, carbon–oxygen bonds, carbon–sulfur bonds, and hydrogen only. The usefulness of measurements of residual amounts of other components of nitrogenase assays is also discussed.