*3.2. Fluorescence Spectroscopic Studies*

Highly sensitive Fluorolog tau3 spectrofluorometer (Jobin-Yvon/SPEX, Longjumeau, France) was used to investigate the fluorescence spectra of the different solutions. For data collection, the photon counting method with 0.1 s integration time was used. Excitation and emission bandwidths were set to 4 nm. A 10 mm thickness of the fluorescent probes with right-angle detection was applied. Temperature-dependent steady-state fluorescence spectroscopic measurements were carried out at different temperatures: 298.2 K, 303.2 K, 308.2 K, and 313.2 K. The fluorescence emission spectra of SMT (30 µM) was recorded in the absence and presence of increasing concentration of BCD or RAMEB (0–3 mM) in different phosphate buffers, using 280 nm excitation wavelength. Similarly, to our previous studies [30,44,45], overall and stepwise association constants of the complex formation were calculated by non-linear fitting, based on the fluorescence emission data obtained, employing the HyperQuad2006 program package [46].

To determine the thermodynamic parameters, temperature dependence of the complex stabilities was examined. According to the van't Hoff Equation (1) the temperature-dependence of the association constants offers possibility to determine the thermodynamic parameters related to the formation of the SMT-BCD and SMT-RAMEB complexes:

$$
\ln \text{K} = -\frac{\Delta \text{G}}{RT} = -\frac{\Delta H}{RT} + \frac{\Delta \text{S}}{R} \text{ \AA} \tag{1}
$$

where the ∆*H* and ∆*S* stand for the enthalpy and entropy changes of the complex formation, while ∆*G* is the Gibbs free energy change. *R* stands for the gas constant, while *T* is the temperature in Kelvin.
