Figure 6.7 Determination of NADH in a cell extract from cultivation of
Applied to a cell extract from a lactic bacteria culture where the NAD* concentration is much higher than
the NADH concentration completely wrong results would be obtained if the NAD* content of the sample
is not quantitatively removed before adding the ADH enzyme to start the experiment. Nordkvist (2001)
has shown that treatment of a sample which originally contains 1 pM NADH and 10 pM NAD* for 10
minutes at 56° C and pH = 12.5 completely destroys the NAD*: The initial slope of the absorbance signal
vs. time is exactly the same as in a sample with only 1 uM NADH.
The assay must be calibrated by measurements of the initial slope of the absorbance signal with known
amounts of NADH added. But the cell extract sample has to undergo a harsh treatment with alkali at
56°C to remove NAD*, and therefore a number of cell extracts are spiked with known amounts of NADH
and incubated at 56°C for 10 minutes. Ethanol, and MTT0X
are added in excess, and adding a sufficient
amount of ADH starts the experiment.
Fig. 6.7 shows that the initial slope of the absorbance vs. time signal is a linear function of the added
NADH (pM). The intersection with the abscissa axis represents the amount of NADH in the assay that
came from the original cell extract.
From Fig. 6.7 one determines the concentration of NADH in the original cell sample withdrawn from the
reactor to be 0.156 pM. The biomass concentration in the reactor was 0.684 g L'1, and consequently the
intracellular NADH concentration is 0.228 pmoles (g DW)'1, or with a cell density of 0.59 g mL ' cell the
molarity of NADH in the cell is calculated to 0.134 mM. In a series of 5 experiments based on samples
withdrawn from the same exponentially growing anaerobic lactic bacteria culture the mean NADH
concentration was found to be 0.172 mM with a standard deviation of 0.029 mM, or 17%._____________