Time Resolved Fluorescence Studies of Genetically Encoded Protein Sensor Frex for Nadh and pH Levels

Abstract

Reduced nicotinamide adenine dinucleotide (NADH) and its oxidized form, NAD+, are the most important coenzymes found in all living cells. For many years, researchers have relied on the weak NADH endogenous fluorescence signal to determine the NADH level. We have recently reported a series of genetically encoded fluorescent sensors highly specific for NADH level1. However, those NADH sensors are also found to be very sensitive to pH value of the local environments, which may result in errors in the analysis of NADH level or NADH/NAD+. We here report that the time resolved fluorescence of NADH sensor Frex can simultaneously characterize the pH and NADH level by using a time correlated single photon counting (TCSPC) technique, whose scale is from 100ps to 30ns. Frex sensors were excited at 420 and 485 nm, respectively. Both the total fluorescence photon counting number N and the fluorescence time distribution have been obtained. All fluorescence transients of Frex sensors reveal complex, i.e. multiexponential behavior. Similar to our previous report1, the N ratio under two excitation wavelengths N485/N420 can be used to characterize the NADH level. Meanwhile, the decay associated spectra (DAS) can be fitted by 3 exponential components decay function. We found that the percentage α of the components with the lifetimes of about 0.2, 1 and 3 ns were proportional to the pH values. Therefore, both the NADH and pH levels can be obtained from time resolved fluorescence spectra. No parallel experiment is needed any more to calibrate the pH effects. Candidate mechanisms and further experiments including femtosecond dynamics will be discussed.Reference:1 Zhao, Y. et al. Genetically Encoded Fluorescent Sensors for Intracellular NADH Detection. Cell Metab14, 555-566, (2011).