*3.1. Intrinsic Fluorescence*

The pattern of changes exhibited in the protein fluorescence after adding a ligand provides information with structure and hence the protein's function [46]. Steady-state fluorescence quenching has been used to determine various binding parameters of ligand– protein interactions. Trp residue has the strongest fluorescence intensity and is the most sensitive to the changes in the micro-environment [47], and its emission wavelength is more sensitive to the microenvironment, indicating the protein conformational changes after binding with drugs [48]. Generally, the quenching mechanism can be classified into three categories: (1) dynamic quenching, which is caused by the collision between molecules in the transition to the excited state; (2) static quenching, which is caused by the formation of a complex between the fluorophore and the quencher; (3) the combined static and dynamic quenching [49]. The quenching was carried out with increasing concentrations of the ligands against α-amylase at different temperatures (Figures 1A and 2A). As indicated by the spectra, quenching was observed with increasing concentration of both the ligands caffeic

and coumaric acid. This decrease in the fluorescence intensity amid the addition of caffeic acid and coumaric acid indicates that the ligands interact in the microenvironment of aromatic residues of α-amylase and mask the internal optimal fluorescence intensity. Previous investigations have shown that many other polyphenols exhibited similar behavior, thereby decreasing the fluorescence intensity of α-amylase [29,37,38]. Fluorescence quenching data were evaluated mathematically employing Stern–Volmer, modified Stern–Volmer, and van't Hoff equations to calculate the binding and thermodynamic parameters [26,28].

**Figure 1.** Binding between caffeic acid and α-amylase. (**A**) Quenching in fluorescence intensity of α-amylase (5 µM) in the absence and presence of varying caffeic acid concentration (0–30 µM) at 298 K, (**B**) Stern–Volmer plot at different temperatures, (**C**) modified Stern–Volmer plot at different temperatures, and (**D**) van't Hoff thermodynamics plot at three different temperatures.

In the SV plot of *F*0*/F* vs. caffeic acid and *F*0*/F* vs. [coumaric acid] (Figures 1B and 2B), the slope gives the value of the Stern–Volmer constant (*K*sv) [40]. Figures 1B and 2B apparently show linear SV plots for caffeic acid–α-amylase and coumaric acid–α-amylase, respectively. Fluorescence quenching can be static or dynamic or a combination of both [34,35]. Temperature dependency of *K*sv reveals the type of quenching operative for a particular interaction, i.e., protein–ligand complex formation is driven by static or dynamic quenching. The *K*sv value decreases with increasing temperature for static quenching due to complex formation, which undergoes dissociation on increasing the temperature. In contrast, the opposite effect occurs for dynamic quenching, where *K*sv increases with temperature. Hence, the values of *K*sv were estimated at three different temperatures for α-amylase-caffeic acid and α-amylase-coumaric acid and are enumerated in Tables 1 and 2, respectively. *K*sv increases with increasing temperature for α-amylase–caffeic acid interaction, implying dynamic quenching. On the contrary, *K*sv values were found to decrease with increasing temperature for α-amylase–coumaric acid, suggesting the presence of static quenching. These observations can corroborate previously reported results [30,40]. Further, the quenching mode was confirmed by finding the biomolecular quenching rate constant (*K*q) using the equation *<sup>K</sup>*<sup>q</sup> <sup>=</sup> *<sup>K</sup>*sv/τ<sup>0</sup> (τ<sup>0</sup> = 2.7 <sup>×</sup> <sup>10</sup>−<sup>9</sup> s). The values of *K*<sup>q</sup> for α-amylase–caffeic acid

(Table 1) and α-amylase–coumaric acid (Table 2) were found to be higher than the maximum dynamic quenching constant (nearly 10<sup>10</sup> M−<sup>1</sup> s −1 ) [49]. Thus, it can be concluded that α-amylase–caffeic acid complex formation was driven by dynamic quenching while a combination of static and dynamic directs α-amylase–coumaric acid complex formation, while α-amylase–coumaric acid interaction was driven by static quenching. Additionally, the binding constant (*K*) was also calculated, revealing the binding affinity for protein (Table 2). Fluorescence quenching data were fitted into a modified Stern–Volmer equation with the intercept of the plot giving the value of binding constant (*K*) for both the ligands (Figures 1C and 2C). It was found to be of the order of 10<sup>4</sup> M−<sup>1</sup> for α-amylase–caffeic acid complex, while for α-amylase–coumaric acid, *K* was of the order of 10<sup>4</sup> M−<sup>1</sup> , but lesser than caffeic acid, suggesting that caffeic acid binds to α-amylase with a higher affinity as compared to coumaric acid. Table 1 depicts the values of *K* at different temperatures for α-amylase–caffeic acid complex, which was found to decrease with increasing temperature, implying that a more stable complex is formed at lower temperatures. Table 2 depicts the values of K obtained for α-amylase–coumaric acid complex.

**Figure 2.** (**A**) Binding between p-coumaric acid and α-amylase. Fluorescence quenching intensity of α-amylase (5 µM) in the absence and presence of varying p-coumaric acid concentration (0–30 µM) at 298 K, (**B**) Stern–Volmer plot at different temperatures, (**C**) modified Stern–Volmer plot at different temperatures and (**D**) van't Hoff thermodynamics plot at three different temperatures.


**Table 1.** Binding and thermodynamic parameters obtained for caffeic acid–α- amylase interaction obtained through fluorescence spectroscopy.

**Table 2.** Binding and thermodynamic parameters obtained for coumaric acid–α- amylase interaction obtained through fluorescence spectroscopy.


The data obtained at different temperatures are fitted into the van't Hoff equation to find the thermodynamic parameters of the system for both the ligands (Figures 1D and 2D). The slope of this plot gives −∆*H*/R, and the intercept gives the value of ∆*S*/R. The magnitude and the sign of the thermodynamic parameters (∆*H*, ∆*S* and ∆*G*) offer a clue about the forces that drive the reaction. Table 1 shows the various thermodynamic parameters obtained for the α-amylase–caffeic acid system, while Table 2 shows the thermodynamic parameters obtained for the α-amylase–coumaric acid system. We obtained negative ∆*H* and ∆*S* for the α-amylase–caffeic acid system, revealing the existence of van der Waals force and hydrogen bonding, while positive ∆*H* and ∆*S* were obtained for α-amylase–coumaric acid, implying the reaction to be driven predominantly by hydrophobic interaction [50,51]. Additionally, negative ∆G for both the systems suggested the spontaneous nature of the reaction.
