FIGURE 3.

dl-Lipoamide equilibrium fluorescence titration of pig heart E3. A, pig heart E3 fluorescence was excited at 280 nm and titrated with dl-lipoamide in conditions described under “Experimental Procedures.” Fluorescence spectra from 294 to 400 nm decreased with increasing dl-lipoamide additions (0, 0.016, 0.094, 0.373, 0.74, 1.43, 2.66, 4.53, 6.6, and 9.8 mm) (black to blue lines). These data (294–400 nm) were corrected (dashed lines in A) for an inner filter effect due to the absorption of dl-lipoamide as shown in B. Fluorescence spectra (A) from ∼400 to 500 nm increased with increasing dl-lipoamide additions (black to green). A fluorescence intensity peak was also observed near 500 nm and remained constant in intensity throughout the titration (black to yellow). B, absorption spectra of dl-lipoamide with increasing concentrations (0.526, 1, 1.98, 2.66, 3.62, 5.4, 7.72, and 9.97 mm; black to blue solid lines). Inset, dl-lipoamide absorption spectra were fitted (dashed lines) using linear least squares to obtain the molar absorptivity (m⁻¹ cm⁻¹) of dl-lipoamide to be used for inner filter effect correction. C, α values are plotted as a function of total dl-lipoamide. D, increasing concentrations of dl-lipoamide (0, 0.0157, 0.0938, 0.373, 0.74, 1.427, 2.66, and 4.53 mm; black to green) in the absence of protein were excited at 330 nm. E, pig heart E3 FAD fluorescence was excited at 455 nm and titrated with dl-lipoamide in the same conditions in A. Pig heart E3 FAD fluorescence spectra are shown with increasing dl-lipoamide concentrations (0, 0.015, 0.046, 0.156, 0.373, 1, 2, 3.6, and 5.4 mm; black to yellow).