Monitoring Metabolic Responses in Saccharomyces Cerevisiae using Fluorescence-Based Detection of NADH Conformation

Abstract

Metabolic co-factors such as reduced nicotinamide adenine dinucleotide (NADH) are fluorescent and can be used to monitor cellular metabolism related to respiration and mitochondrial function. While fluorometric monitoring typically focuses on total NADH concentration, it is the free, as opposed to protein-bound, NADH pool that couples the various biochemical reactions (e.g., involving dehydrogenases) and that is the determinant of reaction velocities. Thus the level of free versus protein-bound NADH is an important physiologic parameter for monitoring metabolic responses. This work explores changes in intracellular NADH conformation when S. cerevisiae (baker's yeast) is subject to various metabolic modifiers (including cyanide, FCCP, glucose, and ethanol) and various oxygenation levels. NADH tends to exist in a folded conformation when free and an unfolded conformation when protein bound, with the excited-state emission shifting to shorter wavelengths upon unfolding. Thus we excite intracellular NADH at 337-nm wavelength using a nitrogen laser, recording the emission spectrum in real time using a spectrograph coupled to an intensified CCD detector. Results indicate that when NADH concentration increases or decreases, the proportion of free NADH increases or decreases as well, although there are differences in the time course of the two measures.