*2.4. Fluorescence-Based Binding*

Fluorescence spectroscopy serves as an important technique to provide an insight into the protein–ligand interactions, revealing different binding parameters that give an idea about the strength of interaction between a protein and ligand [32]. Intrinsic fluorescence depicts changes in the local microenvironment of aromatic amino acid residues and this serves as an important technique in determining the protein-ligand complex formation [33,34]. When a decrease in the fluorescence of native protein is observed with increasing concentration of the ligands, it is referred to as fluorescence quenching [35]. Fluorescence quenching was performed at three different temperatures (15, 20, and 25 ◦C). Fluorescence emission spectra of free HSA and HSA with different HpzA concentrations (0–11 µM) at various temperatures are shown in Figure 9A–C. Native HSA showed fluorescence emission maxima at 346 nm. There was a noticeable decrease HSA's fluorescence intensity with increasing HpzA concentration, with no peak shift. Fluorescence quenching can be either static, dynamic, or a combination of both [36]. The deviation of the binding parameters with temperature delineates the operative quenching for a particular interaction. The quenching data obtained were fitted into the Stern–Volmer, modified Stern–Volmer, and van 't Hoff equations to obtain various binding and thermodynamic parameters for HSA–HpzA interaction as per previously published reports [37,38]. Table 1 shows the obtained values of the Stern–Volmer constant (*K*sv) at various temperatures, which were found to increase with increasing temperature, revealing the existence of the dynamic mode. Moreover, to confirm the quenching mode, a biomolecular quenching rate constant (*K*q) was calculated using *<sup>K</sup>*sv <sup>=</sup> *<sup>K</sup>*q/τ<sup>0</sup> (τ<sup>0</sup> = 2.7 <sup>×</sup> <sup>10</sup>−<sup>9</sup> s), and the value was found to be higher than that of the maximum dynamic quenching constant (nearly 10<sup>10</sup> M−<sup>1</sup> s −1 ) [39], confirming the existence of a combination of static and dynamic quenching. Figure 10A shows the experimental fitting obtained in accordance with the modified Stern–Volmer equation. The slope of the plot gives the number of binding sites (*n*) while the intercept gives the binding constant (*K*). HpzA binds to HSA with a high binding affinity, (*<sup>K</sup>* = 9.3 <sup>×</sup> <sup>10</sup><sup>5</sup> <sup>M</sup>−<sup>1</sup> at 25 ◦C). The value of *K* was found to increase at a higher temperature suggesting that the HSA–HpzA complex is more stable at high temperatures. These observations affirm in silico results advocating the significant binding affinity between HpzA and HSA. Thermodynamic parameters were also found for the HSA–HpzA complex fitting the obtained data in the van 't Hoff equation. Figure 10B shows the van 't Hoff plot obtained for the HSA–HpzA complex. Table 1 shows the obtained thermodynamic parameters for the complex. Negative ∆*H* and ∆*S* implied the van der Waals and hydrogen bonding as the dominant forces driving the complex formation. Moreover, a negative ∆*G* implied the spontaneous nature of the reaction. *Molecules* **2021**, *26*, x 9 of 13 confirming the existence of a combination of static and dynamic quenching. Figure 10A shows the experimental fitting obtained in accordance with the modified Stern–Volmer equation. The slope of the plot gives the number of binding sites (*n*) while the intercept gives the binding constant (*K*). HpzA binds to HSA with a high binding affinity, (*K* = 9.3 × 105 M−1 at 25 °C). The value of *K* was found to increase at a higher temperature suggesting that the HSA–HpzA complex is more stable at high temperatures. These observations affirm in silico results advocating the significant binding affinity between HpzA and HSA. Thermodynamic parameters were also found for the HSA–HpzA complex fitting the obtained data in the van 't Hoff equation. Figure 10B shows the van 't Hoff plot obtained for the HSA–HpzA complex. Table 1 shows the obtained thermodynamic parameters for the complex. Negative Δ*H* and Δ*S* implied the van der Waals and hydrogen bonding as the dominant forces driving the complex formation. Moreover, a negative Δ*G* implied the spontaneous nature of the reaction. **Table 1.** Binding and thermodynamic parameters obtained for the HSA–HpzA complex from fluorescence quenching studies. **Temperature (°C)**  *Ksv* **(104 M−1)** *K* **105 M−<sup>1</sup>** *<sup>n</sup>***∆***<sup>G</sup>* **kcal mol<sup>−</sup><sup>1</sup> ∆***S* **cal mol<sup>−</sup>1 K−<sup>1</sup> ∆***H* **kcal mol<sup>−</sup><sup>1</sup>** ∆**, kcal mol<sup>−</sup><sup>1</sup>** 15 1.8 0.36 1.07 −7.78 −165.26 −55.38 −47.59 20 3.33 1.17 1.10 −6.96 −48.42 25 5.19 9.35 1.25 −6.13 −49.24

**Figure 9.** Fluorescence emission spectra of HSA in the absence and presence of varying HpzA concentrations (0–11 μM) at (**A**) 15 °C, (**B**) 20 °C, and (**C**) 25 °C. **Figure 9.** Fluorescence emission spectra of HSA in the absence and presence of varying HpzA concentrations (0–11 µM) at (**A**) 15 ◦C, (**B**) 20 ◦C, and (**C**) 25 ◦C.

**Figure 10.** (**A**) Modified Stern–Volmer plot of the HSA–HpzA complex at different temperatures.

(**B**) van 't Hoff plot of the HSA–HpzA complex.


*Molecules* **2021**, *26*, x 9 of 13

spontaneous nature of the reaction.

*Ksv* **(104 M−1)** *K*

15 1.8 0.36 1.07 −7.78

**105 M−<sup>1</sup>** *<sup>n</sup>***∆***<sup>G</sup>*

rescence quenching studies.

**Temperature (°C)** 

**Table 1.** Binding and thermodynamic parameters obtained for the HSA–HpzA complex from fluorescence quenching studies.

**Table 1.** Binding and thermodynamic parameters obtained for the HSA–HpzA complex from fluo-

**kcal mol<sup>−</sup><sup>1</sup>**

20 3.33 1.17 1.10 −6.96 −48.42 25 5.19 9.35 1.25 −6.13 −49.24

**∆***S* **cal mol<sup>−</sup>1 K−<sup>1</sup>**

−165.26 −55.38

**∆***H* **kcal mol<sup>−</sup><sup>1</sup>**

∆**, kcal mol<sup>−</sup><sup>1</sup>**

−47.59

confirming the existence of a combination of static and dynamic quenching. Figure 10A shows the experimental fitting obtained in accordance with the modified Stern–Volmer equation. The slope of the plot gives the number of binding sites (*n*) while the intercept gives the binding constant (*K*). HpzA binds to HSA with a high binding affinity, (*K* = 9.3 × 105 M−1 at 25 °C). The value of *K* was found to increase at a higher temperature suggesting that the HSA–HpzA complex is more stable at high temperatures. These observations affirm in silico results advocating the significant binding affinity between HpzA and HSA. Thermodynamic parameters were also found for the HSA–HpzA complex fitting the obtained data in the van 't Hoff equation. Figure 10B shows the van 't Hoff plot obtained for the HSA–HpzA complex. Table 1 shows the obtained thermodynamic parameters for the complex. Negative Δ*H* and Δ*S* implied the van der Waals and hydrogen bonding as the dominant forces driving the complex formation. Moreover, a negative Δ*G* implied the

**Figure 10.** (**A**) Modified Stern–Volmer plot of the HSA–HpzA complex at different temperatures. (**B**) van 't Hoff plot of the HSA–HpzA complex. **Figure 10.** (**A**) Modified Stern–Volmer plot of the HSA–HpzA complex at different temperatures. (**B**) van 't Hoff plot of the HSA–HpzA complex.
