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

✍ Scribed by Michael J. Dilworth; Marie E. Eldridge; Robert R. Eady

• Publisher: Elsevier Science
• Year: 1992
• Tongue: English
• Weight: 480 KB
• Volume: 207
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(92)90491-o

✦ Synopsis

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.