Analytical Biochemistry 1992-11-15

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

M J Dilworth, M E Eldridge, R R Eady

Index: Anal. Biochem. 207(1) , 6-10, (1992)

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Abstract

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.