Chemically coupled spectrophotometric assays based on flow injection analysis: Determination of nitrogenase by assays for creatine, ammonia, hydrazine, phosphate, and dithionite
✍ Scribed by Lawrence C. Davis; Gary A. Radke
- Publisher
- Elsevier Science
- Year
- 1984
- Tongue
- English
- Weight
- 702 KB
- Volume
- 140
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Micromethods of direct chemical coupling have been developed for several different enzyme reactions, using the principles of flow injection analysis. Samples of l-25 ~1 are injected into a flowing stream of color-forming reagents and the peak of color change is measured after about 1 min. Alternatively, continuous slow infusion of a reacting system (5-100 pl/min) gives a continuous change of color which can be monitored to derive enzyme reaction rates. These techniques are highly sensitive, requiring a few nanomolcs of the substance being detected. Phosphate, ammonia, dithionite, creatine, and hydrazine have been measured. Consumption of reagents is less than 75 ml per hour; typical sample throughput is 30-40 samples per hour by the injection method, and 5 samples per hour by continuous infusion. The procedure has been applied to nitrogenam, continuously monitoring crcatine produced from crcatine phosphate by creatine kinase which is used to supply a constant level of ATP for nitrogenase. In this way nitrogenase activity can be determined over a wide range of enzyme concentrations. Production of inorganic phosphate directly from ATP, by injection of formaldehydequenched samples, was used when coupling to creatine kinase was not possible. Both injection of aliquots and continuous infusion were used for detection of hydrazine during nitrogenam reduction of azide, and the injection method has been used for ammonia assay during dinitrogen reduction. Dithionite oxidation was measured directly from decolorization of iodine, after trapping both dithionite and bisulfite with formaldehyde.