*3.1. Fluorescence Quenching and Enhancement*

The fluorescence spectra for the protein BSA for the binary and the ternary system were recorded at 280 nm (excitation wavelength) and an emission wavelength of 300–500 nm (Figure 1a–c). Both the ligands ERL and QUR decreased the fluorescence intensity of BSA. At increased ligand concentration ERL and QUR, the fluorescence intensity of BSA reduced further. The reduction in fluorescence intensity of BSA in the presence of QUR was higher than in the presence of ERL. A redshift of 8 nm was observed in the emission wavelength of BSA on interaction with ERL, indicating increased polarity and less hydrophilicity in the aromatic amino acid microenvironment [40–42]. A blue shift was also observed in the fluorescence emission spectra of BSA in the presence of QUR, as reported by earlier studies indicating higher hydrophobicity and a decline in polarity in the microenvironment of aromatic amino acid residues present in BSA [43]. The quenching behavior for the BSA-ERL binary system was determined with the help of the Stern Volmer equation [44]:

$$\frac{F\_0}{F} = \mathbf{1} + \mathbf{K}\_{\rm sv}[\mathbf{Q}] = \mathbf{1} + k\_q \tau\_0[\mathbf{Q}]$$

$$k\_q = \mathbf{K}\_{\rm sv} / \tau\_0 \tag{1}$$

where *F*<sup>0</sup> is BSA's fluorescence intensity, and *F* is BSA's fluorescence intensity in the presence of quencher. Stern Volmer constant is given as *Ksv*, and (Q) is quencher concentration. The *k<sup>q</sup>* biomolecular quenching constant and *τ*<sup>0</sup> the lifetime of the fluorophore in the absence of quencher and is valued at 10−<sup>8</sup> s for biopolymers.

The Stern Volmer plot for the BSA-ERL system is presented in Figure 1d. A decrease in the *Ksv* values was observed with a rise in temperature for the BSA-ERL system (Table 1). Therefore, reducing *Ksv* values with a temperature rise is associated with the static quenching mechanism. In the case of dynamic quenching, there is an increase in the *Ksv* values [15,44]. Thus, a static quenching mechanism for the BSA-ERL system is suggested based on complex formation between BSA and ERL. The BSA-QUR system also follows a static quenching behavior as revealed by earlier studies [43]. The static quenching mechanism for the BSA-ERL system can also be established based on biomolecular quenching constants *<sup>k</sup>q*, which are of the order of 2 <sup>×</sup> <sup>10</sup><sup>10</sup> <sup>L</sup>·mol−<sup>1</sup> s −1 for dynamic quenching and *<sup>k</sup><sup>q</sup>* values higher than the maximum value of 2 <sup>×</sup> <sup>10</sup><sup>10</sup> <sup>L</sup>·mol−<sup>1</sup> s −1 can be attained for biomolecular quenching constants only during a static quenching. Since the *k<sup>q</sup>* value for the BSA-ERL system given in (Table 1) was high, a static quenching mechanism is suggested for this system in accordance with earlier studies [15,44]. The quenching constant at room temperature for the (BSA-QUR)-ERL ternary system is presented in Table 2. The UV absorbance studies for the BSA-ERL system have reported an increase in the absorbance of BSA at 280 nm.

**Table 1.** Stern Volmer *Ksv* and bimolecular quenching constant *kq*.


\* standard deviation.


**Table 2.** Binding parameters binary and ternary systems and thermodynamic parameters for BSA-ERL system.

\* standard deviation.

**Figure 1.** Fluorescence spectra of BSA (1.5 μM) with: ERL (0.00–27.5 μM) (**a**); Fluorescence spectra of BSA (1.5 μM) with QUR (0.00–35 μM) (**b**); Fluorescence spectra of BSA (1.5 μM) with QUR (5.5 μM)-ERL (0.00–27.5 μM) at (λex = 280 nm and λem = 300–500 nm) (**c**); Stern Volmer Plot for BSA(1.5 μM) -ERL (0.00–27.5 μM system at a temperature of 298, 303, 310 K (**d**); double reciprocal plot [(F<sup>0</sup> − F)/F] versus log [Q] for the BSA-ERL system to obtain the binding constant (**e**); van 't Hoff plots to **Figure 1.** The fluorescence spectra for BSA (1.5 µM) with: (**a**) ERL (0.00–27.5 µM); (**b**) QUR (0.00–35 µM); (**c**) QUR (5.5 µM)-ERL (0.00–27.5 µM) at (λex = 280 nm and λem = 300–500 nm); (**d**) Stern Volmer Plot for BSA(1.5 µM) -ERL (0.00–27.5 µM system at a temperature of 298, 303, 310 K; (**e**) double reciprocal plot [(*F*<sup>0</sup> − *F*)/*F*] versus log [*Q*] for the BSA-ERL system to obtain the binding constant (**b**); (**f**) van't Hoff plots to obtain the for thermodynamic parameters for the BSA-ERL system interaction.

obtain the for thermodynamic parameters for the BSA-ERL system interaction (**f**).

The Stern Volmer plot for the BSA-ERL system is presented in Figure 1d. A decrease in the *Ksv* values was observed with a rise in temperature for the BSA-ERL system (Table 1). Therefore, reducing *Ksv* values with a temperature rise is associated with the static

based on complex formation between BSA and ERL. The BSA-QUR system also follows a
