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A) Free NADH in solution is characterized by fluorescence decay times of 390 +/− 20 ps and 1140 +/− 60 ps. (B) Approximately 95% of the total NADH concentration has the shorter lifetime. (C) These characteristics are independent of the NADH concentration. (D) FLIM analysis shows a linear increase in concentration, as expected. These results can be understood by considering that free NADH in solution can fluctuate between an extended and a folded conformation (afterScott et al. (1970).
