FIG. 3.

Peredox protein fluorescence lifetime NAD⁺/NADH dose–response curves. (A) Peredox protein fluorescence lifetime dose–response curve at 25°C. Purified Peredox protein fluorescence lifetime response to increasing ratios of lactate/pyruvate (bottom axis) and corresponding levels of NAD⁺/NADH (top axis). Error bars indicate standard deviation, n = 4 independent protein samples (black circles), and the Hill fit to the grouped average is shown in black. Fits of the individual experiment data sets yielded an average K_R lactate/pyruvate value (ratio giving a half-maximal response) of 20.7 ± 2.0, which corresponds to a K_R NAD⁺/NADH of 529 ± 50. (B) Peredox protein fluorescence lifetime dose–response curve at 35°C. Purified Peredox protein fluorescence lifetime response to increasing ratios of lactate/pyruvate (bottom axis) and corresponding levels of NAD⁺/NADH (top axis). Error bars indicate standard deviation, n = 3 independent protein samples (black circles), and the Hill fit to the grouped average is shown in black. The average K_R lactate/pyruvate value for all experiments was 23.5 ± 4.2, corresponding to a K_R NAD⁺/NADH of 255 ± 45. At elevated temperature, both the dynamic range and the K_R of Peredox increase. (C) pH dependence of the fully NADH and NAD⁺ loaded states of the sensor (purified protein at 34°C, mean ± range, n = 2).