*3.2. Synchronous Fluorescence*

Synchronous fluorescence spectroscopy is used to have deeper insights into the conformational changes in the proteins microenvironment comprising tyrosine and tryptophan residues. Synchronous fluorescence provides information on conformational changes in the molecular environment of fluorophores of proteins once the ligands bind to them [52]. When ∆λ (λem − λex) is kept at 60 nm or 15 nm, the synchronous fluorescence spectra expose the information about the microenvironment of tryptophan and tyrosine residues, respectively. Figure 3 shows the synchronous fluorescence spectra of free α-amylase and α-amylase with varying concentrations of caffeic acid (upper panel) and coumaric acid (lower panel).

**Figure 3.** Synchronous fluorescence spectra for (**A**) Tyrosine residue (∆λ = 15) and (**B**) Tryptophan (∆λ = 60) of α-amylase (4 µM) in the absence and presence of caffeic acid (0–40 µM). Panel (**C**,**D**) are the spectra obtained under similar conditions for p-coumaric acid.

In the case of ∆λ = 15 nm, a shift in the fluorescence emission maxima of α-amylase in the presence of caffeic acid (Figure 3A) and coumaric acid (Figure 3C) implies that the local environment around tyrosine residue changed significantly in the presence of both the ligands. However, for ∆λ = 60 nm, no shift in the emission maxima of α-amylase is observed for both the ligands (Figure 3B,D), suggesting no change in the local environment around tryptophan residues.
