Studies of yeast cell oxygenation and energetics by laser fluorometry of reduced nicotinamide adenine dinucleotide

It is of fundamental importance for biological scientists to assess cellular energetics. Under aerobic conditions, the tricarboxylic acid cycle (TCA cycle) is coupled with the mitochondrial electron cascade pathway to provide the cell with energy. The nicotinamide adenine dinucleotide-conjugated pair (NAD⁺ and NADH) is the coenzyme in numerous important biomedical reactions which include several important dehydrogenase reactions in the TCA cycle. Based on Le Chatelier's principle, NADH will accumulate when this energy production mechanism is impaired. Traditionally, the quantities of NADH and NAD⁺ may be measured by chemical assay methods. We can avoid these tedious analyses by exploiting the significant difference between the ultraviolet absorption spectra of this redox pair. However, because of the opacity of biological samples and the interference of other biochemicals that also absorb ultraviolet radiation, measurement of NADH and NAD⁺ concentrations in vivo by absorption spectroscopy is not feasible. Fluorescence spectroscopy has the advantages of being non-intrusive and highly sensitive. Because the NAD⁺ molecule lacks the absorption of the NADH molecule at a wavelength of 340 nm, NADH molecules can be excited selectively by radiation at this wavelength. Using a laser as the excitation source improves the selectivity and sensitivity of the fluorometric method. Furthermore, the system may be coupled with fiber-optics components and will provide excellent flexibility in future studies.