FIGURE 3.

Emission decays of donor- and donor-acceptor-labeled NBDs. A, effect of ATP on sensitized fluorescein emission decay from MJ-C14. Black trace (Tb only), emission of MJ-C14 labeled with Tb³⁺ only, measured at 490 nm. Blue (No ATP) and red traces (ATP), sensitized fluorescein emission of MJ-C14 labeled with Tb³⁺ and fluorescein, measured at 520 nm in the absence or presence of 2 mm ATP, respectively. Green trace (MJ-CL), sensitized fluorescein emission (measured at 520 nm) of Cys-less protein (MJ-CL) subjected to the Tb³⁺ and fluorescein labeling procedure. When present, ATP concentration was 2 mm. Tb³⁺ only and ATP intensities were normalized to their corresponding values at 1,000 μs, and the No ATP and MJ-CL intensities were normalized to the ATP intensity at 1,000 μs. The traces are representative of 5 similar experiments. B, effect of ATP on sensitized Cy3 emission decay from MJ-C14. Details are as in panel A, but Cy3 was used as acceptor instead of fluorescein, with the sensitized emission measured at 570 nm. C, effect of Mg-ATP on the sensitized fluorescein emission decay of Tb³⁺/fluorescein-labeled MJ-C14. Semilog plot of the sensitized fluorescein decay measured in 2 mm ATP (red) and after the addition of MgCl₂ (green). The decay in the absence ATP (recorded in the same sample at the beginning of the experiment) was subtracted from the ATP and Mg-ATP decays, resulting in traces that can be fit to a single exponential function (black lines over the ATP and Mg-ATP traces). The decay from MJ-C14 labeled with Tb³⁺ alone (black) is also shown. ATP and Tb³⁺ only were normalized to their intensities at 1,000 μs, whereas the Mg-ATP data were normalized to the ATP intensity at 1,000 μs. D, effect of Mg-ATP on the sensitized Cy3 emission decay of Tb³⁺/Cy3-labeled MJ-C14. Details are as in panel C, but Cy3 was used as acceptor instead of fluorescein.