2.2. Steady-State Fluorescence Spectroscopy

Fluorescence experiments were performed on the Lumina (Thermo Fisher Scientific, Waltham, MA, USA) stationary state spectrofluorimeter equipped with a thermal bath and Xenon lamp. A 100-μL quartz cuvette with a 10 × 2 mm optical path was used in the experiments. The widths of the excitation and the emission slits were adjusted to 10 nm. The wavelength of 295 nm was used to excite the single tryptophan residue of IL-1β (Trp120). The emission spectra were obtained in the range from 305 to 570 nm with a resolution of 1.0 ± 5.0 nm. Each emission point collected was the average of 15 accumulations. The software ScanWave was used to collect the measured data.

In the binding equilibrium experiments, aliquots of piperine (increment of 4 μM) were added in IL-1β solution at 4 μM. Measurements were performed at 288, 298, and 308 K. In the interaction density function analysis, small aliquots of piperine (increments of 1 μM) were added to IL-1β solutions at 4 μM, and 8 μM at a fixed temperature (298 K). In all experiments, the final volume of methanol in the buffer was less than 1.0%.

The correction of the inner filter effects was done with Equation (1), where F_corr and F_obs are corrected and observed fluorescence intensities, and A_ex and A_em are the absorbance at the excitation and the emission wavelengths, respectively, considering a cuvette of 10 × 10 mm of optical path [8].

The Stern–Volmer constant (K_SV) and bimolecular constant (k_q) were obtained from Equation (2)

where F is the observed fluorescence intensity, F₀ is the fluorescence intensity in the absence of piperine and τ₀ is the Trp120 lifetime in the absence of piperine.