Emission Properties of NADH. Studies of Fluorescence Lifetimes and Quantum Efficiencies of NADH, AcPyADH, and Simplified Synthetic Models
Scott, TG; Spencer, RD; Leonard, NJ; Weber, G
2 The fluorescence lifetimes τ and quantum efficiencies q of NADH, AcPyADH, and of the model compounds Ad-C3-NicH and Ad-C6-NicH have been measured in water and in 1,2-propanediol solution in the temperature range 0–30°. For Ad-C3-NicH, NADH, and Ad-C6-NicH at 25° in water the absolute quantum efficiencies are 0.035, 0.019, and 0.017, respectively, to a precision of a few per cent, and the lifetimes are 0.70, 0.40, and 0.28 ± 0.03 to 0.05 nsec The contribution of the ground to lowest singlet transition to the absorption spectrum has been evaluated by fluorescence polarization observations, and from these and the molecular fluorescence spectra, emissive lifetimes Te have been calculated by the equation of Strickler and Berg. For NADH and AcPyADH under various conditions, the relation r= req is followed rigorously over a thirtyfold change in quantum efficiency. The absolute efficiency of quinine sulfate measured either by comparison with these derivatives or by the relation q = τ/τe turns out to be 0.70 ± 0.02 rather than the often quoted 0.55. The observed quantum efficiencies of energy transfer from the adenine to the dihydronicotinamide moiety were as follows for Ad-C3-NicH, NADH, and Ad-C6-NicH at 25° in aqueous solution: 0.44, 0.34, and 0.10, respectively. For these three compounds, comparison of the rates and of the energies of activation for radiationless transitions calculated from the temperature dependence of the lifetimes shows that the quenching processes are essentially identical in 1,2-propanediol but different in aqueous solution, indicating that interactions in water, but not in 1,2-propanediol, are characteristic and specific for each compound. The simplified spectroscopic models, Ad-C3-NicH and Ad-C6-NicH, which were designed to incorporate the absorption–emission chromophores of NADH by linking the adenyl and dihydronicotinamide moieties of NADH with triand hexamethylene chains, were prepared by dithionite reduction of the corresponding precursors possessing the nicotinamide ring in oxidized form. 1970, American Chemical Society. All rights reserved.