Figure 2
Fluorescence and Ca2+ binding of NCaBD and +1EF-2EF. A solution containing 4 µM NCaBD was excited at 280 nm and the emission spectra was recorded at 20°C in the presence of 500 µM EDTA (dotted line) and 100 µM Ca2+ (solid line) (upper panel in A). Solutions of different Ca2+ concentrations were titrated to a solution containing 2 μM NCaBD and the emission intensities were recorded at 350 nm. The normalized fluorescence was fitted with a Hill equation with n = 0.91 (lower panel in A). Ca2+ binding to +1EF-2EF was monitored by fluorescence with an excitation at 280 nm. The intensity enhanced with 1 mM Ca2+ interval and leveled off at 3-4 mM Ca2+ (upper panel in B). The titration monitored at the emission intensity of λ350 nm fit a model with a Hill equation (lower panel in B). Data shown are representative of at least four independent experiments.