Fig. 3. ANS fluorescence spectra in the presence of the F34W/W140F mutant and wild type SNase under different conditions. Each sample contained 60 mM ANS and 5 mM protein in 30 mM Tris-HCl, pH 7.4. The spectra were measured at 25°C. The excitation wavelength was 345 nm and the slit width was 10 nm. From top to bottom: F34W/W140F mutant alone; F34W/W140F mutant with 60 mM pdTp and 10 mM Ca²⁺; wild type SNase alone; wild type SNase with 60 mM pdTp and 10 mM Ca²⁺ (dotted line) and 60 mM ANS itself.