New approaches to studying redox metabolism using time-resolved NAD(P)H fluorescence and anisotropy

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Living cells require the constant input of energy to maintain their characteristic order. This is provided by the chemical reactions of metabolism. A vast number of these reactions are "redox" (reduction-oxidation) reactions, in which electrons are transferred from one molecule to another. During the breakdown of nutrient molecules to extract energy from food, bonds are broken and electrons are passed to a carrier molecule known as NADH. Removal of electrons is referred to as oxidation, and their addition is known as reduction. The metabolism of food therefore involves the oxidation of sugars, proteins and fats and the simultaneous reduction of NADH.The energy passed to NADH during its reduction is converted into a useable form, the cell's "energy currency" adenosine triphosphate (ATP), inside the mitochondria. This is achieved by using the electrons carried by NADH to reduce oxygen to water. This is the fate of almost 100% of the oxygen consumed by the body, and the energy released is stored as ATP. Defects in this process cause the production of reactive oxygen species (ROS). These are highly damaging molecules, and so the cell possesses specific defence mechanisms against their deleterious effects. These defences are maintained by another electron carrier molecule known as NADPH.The antioxidant systems supported by NADPH act to neutralise the harmful ROS produced during the energy generating processes regulated by NADH. These reactions are collectively known as redox metabolism. Imbalance between the two processes is a known factor in the development of a wide range of diseases, including cancer, diabetes and neurodegenerative disorders. In order to make progress in understanding how these diseases occur and testing potential treatments, it is crucial that biomedical researchers are provided with highly accurate tools to investigate redox metabolism in cells and tissues.As a collection of scientists whose expertise includes the use of lasers to study the dynamic behaviour of molecules and the application of these methods to investigate metabolic processes in living tissues, we propose to develop the next generation of approaches to studying the role of redox metabolism in health and disease. We will exploit the intrinsic fluorescence of NADH and NADPH to construct our new experimental technique. While the two molecules emit light of the same colour, the two independent sets of enzymes that they bind to in order to perform their distinct roles will cause contrasting effects on other characteristics of their fluorescence.We will first prepare solutions of NADH and NADPH bound to their respective enzymes to investigate the resulting properties of their fluorescence. The distinct binding sites to which these molecules attach may cause contrasting effects on the time taken for fluorescence to emerge following absorption of a laser pulse, the so-called fluorescence lifetime. Different forces acting on the molecules in the binding site will also cause differences in the freedom of their motion, which can be detected by measuring the rate of change of the polarisation of the light emitted with respect to the light absorbed, the so-called time-resolved fluorescence anisotropy.Following characterisation of the differences in NADH and NADPH fluorescence when bound to their separate sets of enzymes, we will construct a microscope in which these properties can be detected inside living tissues. Inside the complex environment of the cell, the fluorescence signals would be expected to arise from a mixture of NADH and NADPH, both bound to their enzymes and free. We will develop approaches to extract information from these signals, allowing the function of the separate redox pathways that they are involved in to be investigated. As redox metabolism is being found to play a role in an ever growing range of processes, these new approaches will play a key role in enhancing our fundamental understanding of biology.