Table 1.

Fluorescence Lifetime Properties of Peredox^a and Dye Standards

	τ1	f1	τ2	f2		τEmpirical
Peredox NADH, 25°C	1.38 ± 0.15	0.18 ± 0.03	3.22 ± 0.05	0.82 ± 0.03	2.63 ± 0.01	2.63 ± 0.01
Peredox NAD+, 25°C	0.99 ± 0.02	0.58 ± 0.04	2.49 ± 0.06	0.42 ± 0.04	1.87 ± 0.02	1.87 ± 0.02
Peredox NADH, 35°C	1.00 ± 0.09	0.29 ± 0.06	3.19 ± 0.11	0.71 ± 0.06	2.53 ± 0.01	2.53 ± 0.01
Peredox NAD+, 35°C	0.84 ± 0.02	0.65 ± 0.03	2.28 ± 0.09	0.35 ± 0.03	1.65 ± 0.01	1.66 ± 0.01
Fluoresceinb	3.95 ± 0.05	1			3.08 ± 0.01	3.08 ± 0.004
Rhodamine Bc	2.45 ± 0.02	1			2.27 ± 0.01	2.32 ± 0.005

^aAll lifetime values in ns. The values are derived from two-exponential fits of the form f₁ e^−t/τ1 + f₂ e^−t/τ2, convolved with a Gaussian of FWHM ≈140 ps. The summary values for the lifetime are as follows:

, the average lifetime value of the fit function over the measurement range (a window of ∼10 ns); τ_Empirical, the average arrival time of the detected photons, over the measurement range. Protein was in MOPS buffer at pH 7.3, at 25°C. Excitation wavelength was 800 nm, and emission light collected from 500 to 550 nm. Expressed as mean ± SD, n = 4 for all measurements.

^bIn 0.1 M sodium hydroxide, 5 μM. Excitation wavelength 800 nm, emission light collected from 500 to 550 nm. Expressed as mean ± SD, n = 3. Compare to reference fluorescein fluorescence lifetime value of 4.1 ± 0.1 ns (18, 21).

^cIn methanol, 5 μM. Excitation wavelength 800 nm, emission light collected from 603 to 678 nm. Expressed as mean ± SD, n = 3. Compare to reference rhodamine B fluorescence lifetime value of 2.5 ± 0.1 (5).