Effect Of Metal Ions On Fluorescence Kinetics Of Coenzyme NADH

Dihydronicotinamide adenine dinucleotide(NADH)and its oxidation state(NAD⁺)are important coenzymes commonly found in eukaryotes and prokaryotes,participating in biological redox processes and playing a key role in cellular substance metabolism as well as in energy metabolism.NADH is composed of nicotinamide,adenine,and two ribose rings linked by pyrophosphate bridges,and is usually present in aqueous solution,and in a dynamic equilibrium with both a free folded conformation and an unfolded conformation in proteins.In living organisms,some metal ions have a very important role in the enzymatic reactions in which NADH is involved.Among them,the aluminum ion(Al³⁺)in vitro and the magnesium ion(Mg²⁺)in vivo both have good affinity for biophosphates.In aqueous solution,Al³⁺and Mg²⁺preferentially coordinate the two oxygen atoms on the hydroxyl group of the NADH pyrophosphate bridge in the form of a double-coordinated monodentate,generating two strong ionic bonds,which in turn affect the biochemical properties associated with NADH.In this thesis,the effects of metal ions on the intrinsic fluorescence spectra and molecular conformational changes of NADH in aqueous solution were investigated using UV-Vis absorption spectroscopy and steady-state fluorescence spectroscopy in combination with time-correlated single photon counting technique(TCSPC).The main research work consists of the following two parts:1.The interaction of Al³⁺with NADH molecules in aqueous solution was investigated.It was demonstrated that the NADH molecule and Al³⁺would combine at a concentration ratio of 1:2,and the combination would cause the conformational change of the NADH molecule from the unfolded state to the folded state,leading to an increase of the NADH intrinsic fluorescence lifetime and thus the fluorescence enhancement.Meanwhile,the two main conformational forms of NADH in solution were characterized for the first time by using the study of NADH intrinsic fluorescence lifetime amplitude ratio.2.The interaction of magnesium ions with NADH molecules was investigated.The results showed that the NADH molecule would bind with Mg²⁺in aqueous solution mainly at a concentration ratio of 1:1,and the binding formed a stable non-luminescent complex,which lead to static quenching of the intrinsic fluorescence of NADH,resulting in a diminished fluorescence.Subsequently,Mg²⁺was found to have no effect on the conformational changes of NADH molecules in two different solvent environments which were methanol and ethanol.The research work in this thesis does not only contributes to further understanding of the role played by metal ions in the catalytic reactions of biological enzymes,but also allows the effective application of endogenous fluorophore molecules in living organisms to the detection of metal ion concentrations in various production activities.Therefore,it is of great biological significance and instructive value to study the effect of metal ions on the fluorescence kinetics of NADH molecules.