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 (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.
Dilworth
M J
MJ
School of Biological and Environmental Sciences, Murdoch University, Western Australia.
Eldridge
M E
ME
Eady
R R
RR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000641:Ammonia / Q000032:analysis*; D000837:Anion Exchange Resins; D003124:Colorimetry / Q000379:methods; D004396:Coloring Agents; D003401:Creatine / Q000032:analysis*; D004492:Edetic Acid; D006997:Hypochlorous Acid; D007700:Kinetics; D009584:Nitrogen / Q000378:metabolism; D009591:Nitrogenase / Q000032:analysis*; D010084:Oxidation-Reduction; D019800:Phenol; D010636:Phenols; D010725:Phosphocreatine / Q000378:metabolism; D015203:Reproducibility of Results; D013696:Temperature; D014639:Vanadium
D000837:Anion Exchange Resins; D004396:Coloring Agents; D010636:Phenols; D014639:Vanadium; D010725:Phosphocreatine; D019800:Phenol; C034202:iodophenol blue; D006997:Hypochlorous Acid; D000641:Ammonia; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D009591:Nitrogenase; D003401:Creatine; D009584:Nitrogen