*3.6. ANS (8-Anilinonapthalene-1-Sulfonic Acid) Binding Measurements*

The ANS fluorescence measurements were carried out using a Jasco spectrofluorometer (FP6200) at 25 ± 1 ◦C with a 1 cm path length quartz cuvette. The 1:20 ratio for protein to dye at different pH values was used to know the exposure of hydrophobic surfaces. The excitation and emission slits widths were set at 5 nm with a scanning speed of 125 nm min−<sup>1</sup> . The sample was incubated in the dark for 30 min before performing the measurements. The ANS emission spectra were recorded in the range of 400–630 nm with an excitation wavelength of 380 nm.
