Correction for creatine interference with the direct indophenol measurement of NH3 in steady-state nitrogenase assays

Creatine was identified as a major source of interference with the direct phenol/hypochlorite colorimetric determination of ammonia in nitrogenase reaction mixtures. A method is described for removing other compounds which inhibit color development and for compensating for the interference produced by creatine. This method avoids time-consuming microdiffusion and also routinely makes available the efficiency of ATP hydrolysis coupled to substrate reduction (View the MathML source ratio) with N2 as a reducible substrate. Using this method we determined values for this ratio at 30°C of 4.87 ± 0.03 during the reduction of protons to H2 and 7.16 ± 0.14 during the reduction of N2 by the vanadium-containing nitrogenase of Azotobacter chroococcum