nitrogenase and iodophenol-blue

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 (ATP/2e ratio) with N2 as a reducible substrate. Using this method we determined values for this ratio at 30 degrees 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.

Topics: Adenosine Triphosphate; Ammonia; Anion Exchange Resins; Colorimetry; Coloring Agents; Creatine; Edetic Acid; Hypochlorous Acid; Kinetics; Nitrogen; Nitrogenase; Oxidation-Reduction; Phenol; Phenols; Phosphocreatine; Reproducibility of Results; Temperature; Vanadium