NADH calibration curve obtained by UV-Visible spectrophotometry

NADH is an important cofactor in
many redox-dependent reactions in the cell, without which, oxidoreductase could
not function, and there would not be product formation. An intracellular
molecule, and relatively unstable outside of the cellular milieu,
quantification of NADH and characterisation of reaction progress has met with
significant challenges over the past several decades. Recent advances in
development of NADH biosensor offers hope for the fluorescence based detection
of NADH in live cells, thereby, affording a methodology for accurate, real-time
monitoring of reaction progress in living systems. But, NADH biosensor requires
intracellular heterologous expression of biosensor genes, and is technically
complex with additional complications coming from perturbation of cellular
function from biosensor. An alternative simpler approach is the lysis of whole
cells, and UV-Visible spectrophotometry detection of NADH in the whole cell
lysate at 340 nm. This work presents the calibration curve of NADH detected by
UV-Visible spectrophotometry at 340 nm at 1 mL scale in a polystyrene cuvette with
pathlength 10 mm. Results indicate the viability of the approach, particularly
that which emanates from observation of a linear calibration curve with high
correlation coefficient (r2 = 0.9999) between 0 and 10 mM NADH dissolved in deionized water. Collectively, successful generation of a linear
calibration curve for NADH in deionized water within 0 to 10 mM concentration
range suggests the possibility of using UV-Visible spectrophotometry analysis
for detecting intracellular NADH in whole cell lysates. The approach is simple
in concept, and easy to implement, and should find use in a variety of
workflows in biocatalysis and metabolic engineering requiring quantification of
the redox cofactor.