*3.6. Three Dimensional (3D) Fluorescence Spectroscopy*

The 3D fluorescence spectroscopy provides information about protein structural alteration on interaction with a ligand [41]. The 3D fluorescence spectra were accessed for the protein BSA and the protein–ligand BSA-ERL system (Figure 4a,b). Four peaks observed in the 3D spectra were identified as Peak a and b representing Rayleigh scattering (λex = λem) and IInd order scattering peak (λem = 2ex). Higher intensity and scattering were observed for Peak a in the BSA-ERL system compared to BSA since the BSA-ERL complex formed a bigger macromolecule than BSA. The other peaks, Peak I representing

Trp and Tyr fluorophore residues and Peak II (polypeptide backbone structures), were evaluated in the 3D spectra. The intensity of both peaks, Peak I and Peak II, declined in the BSA-ERL protein–ligand system compared to the protein BSA. Hence, it is concluded that the interaction of ERL with BSA altered the microenvironment of the fluorophore residues, which led to these changes in the 3D spectra. Hence it was concluded that structural changes might have occurred in BSA on interaction with ERL. Tyr fluorophore residues and Peak II (polypeptide backbone structures), were evaluated in the 3D spectra. The intensity of both peaks, Peak I and Peak II, declined in the BSA-ERL protein–ligand system compared to the protein BSA. Hence, it is concluded that the interaction of ERL with BSA altered the microenvironment of the fluorophore residues, which led to these changes in the 3D spectra. Hence it was concluded that structural changes might have occurred in BSA on interaction with ERL.

*Molecules* **2022**, *27*, x FOR PEER REVIEW 10 of 16

it was different from both the binary system spectra (Figure 3b).

*3.5. Synchronous Fluorescence Spectroscopic Studies* 

*3.6. Three Dimensional (3D) Fluorescence Spectroscopy* 

from the QUR absorption spectra.

Spectra for BSA was compared to spectra of the studied ligands ERL and QUR, BSA-ERL complex, and BSA-QUR complex. An increase in absorption spectra was observed for the BSA-ERL system compared to BSA alone. The ERL showed different absorption peaks compared to both BSA and BSA-ERL systems. The higher absorption peak for the BSA-QUR system was observed compared to BSA alone, and the spectra were different

The absorption spectra for the ternary system (BSA-QUR)-ERL was also studied, and

The microenvironment alterations in the fluorophore residues can be identified with synchronous fluorescence spectroscopy. The changes in emission wavelength indicate polarity change in the fluorophore residue microenvironment [52]. The synchronous spectra recorded at ∆λ =15 nm and ∆λ =60 nm provide information about the Tyr and Trp residue microenvironment. The spectra for Tyr showed no shift in the emission wavelength, whereas the spectra for Trp showed a slight 1 nm shift. A blue-shift at ∆λ = 60 nm suggests micro-environmental modifications in the vicinity of Trp residue. Further, it is recommended that the BSA experienced an increased hydrophobicity on interaction with ERL.

The 3D fluorescence spectroscopy provides information about protein structural alteration on interaction with a ligand [41]. The 3D fluorescence spectra were accessed for the protein BSA and the protein–ligand BSA-ERL system (Figure 4a,b). Four peaks observed in the 3D spectra were identified as Peak a and b representing Rayleigh scattering (λex = λem) and IInd order scattering peak (λem = 2ex). Higher intensity and scattering were observed for Peak a in the BSA-ERL system compared to BSA since the BSA-ERL complex formed a bigger macromolecule than BSA. The other peaks, Peak I representing Trp and

**Figure 4. Figure 4.**  Three-dimensional fluorescence spectra for BSA ( Three-dimensional fluorescence spectra for BSA (**aa** ) and BSA-ERL system ( ) and BSA-ERL system (**b b** ). ).
