Summary
Incubations of freshly dissolved diaphorase with reduced methylviologen show in the first hours variable but usually rather low activities for the reduction of NAD with reduced methylviologen, as compared to lipoamide dehydrogenase activity at the expense of NADH. However, the former activity increases in a few hours by factors of 5–10 and is then very stable under operational conditions. The half life in the presence of reduced methylviologen at 35°C is >40 days, whereas the lipoamide dehydrogenase activity has a half life of only about 4 h. Even in a rigorously stirred electro-chemical cell the methylviologen dependent NAD reductase activity is very stable.
The enzyme is also suitable for the regeneration of NAD if carbamoylmethylviologen is used. This mediator has a 145 mV less negative redox potential than methylviologen. Again the stability of the enzyme is rather high. Under operational conditions the activity increases for about 9 days and then stays constant for at least 11 days.
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References
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Günther, H., Simon, H. The use of pig heart dihydrolipoamide dehydrogenase (diaphorase) for the regeneration of NADH or NAD. Appl Microbiol Biotechnol 26, 9–12 (1987). https://doi.org/10.1007/BF00282142
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DOI: https://doi.org/10.1007/BF00282142