Macromolecular Crowding Effects on Cellular NADH-enzyme Binding Kinetics

Abstract

Reduced nicotinamide adenine dinucleotide (NADH) is a major cofactor for a large number of biological enzymes that are essential in a myriad of metabolic pathways such as glycolytic and oxidative phosphorylation pathways. In addition, NADH is intrinsically fluorescent and therefore has the potential of serving as a biomarker to monitor mitochondrial dysfunctions associated with aging, cancer, and apoptosis. In this thesis, we investigate how macromolecular crowding may affect the biochemical reaction kinetics of NADH interaction with lactate dehydrogenase (LDH) as a model system in biomimetic crowding (e.g., Ficoll-enriched buffer at 0 ̶ 400 g/L). Using noninvasive, quantitative two-photon fluorescence lifetime and associated anisotropy, we exploit the sensitivities of NADH fluorescence lifetime and rotational diffusion to protein binding. To differentiate between viscosity and crowding effects on the reaction kinetics, we also conducted complementary measurements in glycerol-enriched buffer. Additionally, we are investigating the sensitivity of cellular NADH interaction with dehydrogenases to metabolic manipulations. Our quantitative and non-invasive methodology complements the traditional biochemical and thermodynamics techniques without the destruction of live cells. Intracellular NADH also exists as a mixture of free and enzyme-bound populations at dynamic equilibrium throughout living cells, which can be imaged using fluorescence lifetime imaging for both quantitative and noninvasive assessment of cellular metabolism. 2P-fluorescence lifetime imaging microscopy (FLIM) and 2P- fluorescence anisotropy of intrinsic NADH was measured in cultured mouse embryonic cells under both resting conditions and metabolic-manipulation.