Determination of the tryptophan fluorescence spectra and quenching by acrylamide

The fluorescence spectra of the purified protein samples were collected at 30°C using an F-4500 Hitachi spectrofluorimeter (Hitachi, Tokyo, Japan). Samples were excited at 295 nm, and the emission spectra were recorded in the range of 305 to 400 nm. The scanning speed was 12 nm/min, the slit widths were set to 5 nm (emission and excitation), and the integration time was 1 s. Fluorescence quenching was performed using different concentrations of acrylamide prepared in 10 mM sodium phosphate buffer, pH 6.0. The quenching effect was evaluated by plotting the ratio of the maximum fluorescence (F) in the presence of acrylamide to the maximum fluorescence in the absence (F₀) of acrylamide versus the acrylamide concentration. The Stern-Volmer constant (K_sv), which indicates the extent of tryptophan exposure to the solvent, was calculated from the slopes of the F/F₀ versus [acrylamide] graphs [25, 26].