| Abstract: | This study describes the resazurin/diaphorase system and its use in a kinetic assay for the determination of dehydrogenase activity. This has increased the sensitivity (limit of detection is 2 × 10−5 U/ml) and produced calibration curves which have an extended linear range and better separation of points. The specific activity of diaphorase when resazurin (6.7 μM) is used as a substrate is only about 2 × 10−3 that when 2,6-dichlorophenol-indophenol (DCPIP)2 is used as a substrate. The specific activity of diaphorase can be increased by using higher concentrations of resazurin. The concentration chosen is influenced by the size of fluorometric cuvette used. In a 3.0-ml cuvette, concentrations above 13.4 μM result in lower signals due to the quenching of the fluorescence by resazurin. In a 1.0-ml cuvette, the quenching effect is less severe and a higher concentration (34 μM) of resazurin can be used. Diaphorase activity is first order in the concentration range up to 34 μM of resazurin, and the Km could not be calculated from the range tested. The Km of diaphorase with respect to NADPH is dependent on the concentration of resazurin used. It is 0.78 (± 0.04) and 1.31 (± 0.04) μM at resazurin concentrations of 6.7 and 34 μM, respectively. |
