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.
