*2.2. Fluorescence Emission Spectroscopy of the HSA-QTP Complex*

*2.2. Fluorescence Emission Spectroscopy of the HSA-QTP Complex*  Fluorescence emission spectroscopy is a multipurpose biophysical technique used to study the binding mechanism of protein-ligand interactions and to evaluate the binding parameters [10,12,26]. The fluorescence emission spectra of HSA alone and the HSA-QTP complex are given in Figure 3A. It is apparent from Figure 3A that HSA exhibits a strong emission peak at 340 nm upon excitation at 295 nm due to W-214 residue. Further, the addition of different concentrations of QTP (0–35 μM) leads to the quenching of HSA fluorescence intensity without changing the peak shape. This fluorescence quenching suggests the formation of the HSA-QTP system and suggests a possible microenvironmental alteration in HSA upon treatment with QTP [33,34]. Fluorescence emission spectroscopy is a multipurpose biophysical technique used to study the binding mechanism of protein-ligand interactions and to evaluate the binding parameters [10,12,26]. The fluorescence emission spectra of HSA alone and the HSA-QTP complex are given in Figure 3A. It is apparent from Figure 3A that HSA exhibits a strong emission peak at 340 nm upon excitation at 295 nm due to W-214 residue. Further, the addition of different concentrations of QTP (0–35 µM) leads to the quenching of HSA fluorescence intensity without changing the peak shape. This fluorescence quenching suggests the formation of the HSA-QTP system and suggests a possible microenvironmental alteration in HSA upon treatment with QTP [33,34]. *Molecules* **2022**, *27*, x FOR PEER REVIEW 4 of 17

> **Figure 3.** (**A**) Steady-state fluorescence emission spectra of HSA were recorded in the absence and presence of increasing concentrations of QTP. The intrinsic fluorescence of the HSA was measured at 295 K in the wavelength range of 300–420 nm after exciting at 295 nm. The black arrow represents fluorescence quenching of HSA on titration with QTP (**B**) Stern-Volmer plot for QTP-HSA interaction (295, 300, 310 K). (**C**) Double log plot for the QTP-HSA interaction at different temperatures (295, 300, 310 K). (**D**) van 't Hoff plot (lnK vs. 1/T) for the binding of QTP to HSA. The concentration

of HSA was 5 μM and was titrated with QTP (0–35 μM) in all the experiments (**A**–**D**)**.**

System

2.2.1. Fluorescence Quenching Mechanism (FQM) of the Interactions of the HSA-QTP

According to the literature, the protein's fluorescence quenching mechanism (FQM) consists mainly of dynamic quenching and static quenching. In the case of dynamic quenching, the interaction of the fluorophore with the quencher is indirect. In contrast, in the case of static quenching, a ground state complex formation exists between the fluorophore and quencher [30]. Therefore, the FQM can be sorted out based on their temperature dependence. Furthermore, in the case of static quenching, Ksv values are inversely proportional to temperature, whereas in dynamic Ksv, the values are directly proportional to temperature. Therefore, the FQM of the HSA-QTP system was evaluated by recording the fluorescence spectra of HSA-QTP at different temperatures (295, 300, and 305 K), and the fluorescence quenching data of the HSA-QTP system was analyzed using the Stern-

**Figure 3.** (**A**) Steady-state fluorescence emission spectra of HSA were recorded in the absence and presence of increasing concentrations of QTP. The intrinsic fluorescence of the HSA was measured at 295 K in the wavelength range of 300–420 nm after exciting at 295 nm. The black arrow represents fluorescence quenching of HSA on titration with QTP (**B**) Stern-Volmer plot for QTP-HSA interaction (295, 300, 310 K). (**C**) Double log plot for the QTP-HSA interaction at different temperatures (295, 300, 310 K). (**D**) van 't Hoff plot (lnK vs. 1/T) for the binding of QTP to HSA. The concentration of HSA was 5 μM and was titrated with QTP (0–35 μM) in all the experiments (**A**–**D**)**. Figure 3.** (**A**) Steady-state fluorescence emission spectra of HSA were recorded in the absence and presence of increasing concentrations of QTP. The intrinsic fluorescence of the HSA was measured at 295 K in the wavelength range of 300–420 nm after exciting at 295 nm. The black arrow represents fluorescence quenching of HSA on titration with QTP (**B**) Stern-Volmer plot for QTP-HSA interaction (295, 300, 310 K). (**C**) Double log plot for the QTP-HSA interaction at different temperatures (295, 300, 310 K). (**D**) van 't Hoff plot (lnK vs. 1/T) for the binding of QTP to HSA. The concentration of HSA was 5 µM and was titrated with QTP (0–35 µM) in all the experiments (**A**–**D**).

#### 2.2.1. Fluorescence Quenching Mechanism (FQM) of the Interactions of the HSA-QTP 2.2.1. Fluorescence Quenching Mechanism (FQM) of the Interactions of the HSA-QTP System

System According to the literature, the protein's fluorescence quenching mechanism (FQM) consists mainly of dynamic quenching and static quenching. In the case of dynamic quenching, the interaction of the fluorophore with the quencher is indirect. In contrast, in the case of static quenching, a ground state complex formation exists between the fluorophore and quencher [30]. Therefore, the FQM can be sorted out based on their temperature dependence. Furthermore, in the case of static quenching, Ksv values are inversely proportional to temperature, whereas in dynamic Ksv, the values are directly proportional to temperature. Therefore, the FQM of the HSA-QTP system was evaluated by recording the fluorescence spectra of HSA-QTP at different temperatures (295, 300, and 305 K), and the fluorescence quenching data of the HSA-QTP system was analyzed using the Stern-According to the literature, the protein's fluorescence quenching mechanism (FQM) consists mainly of dynamic quenching and static quenching. In the case of dynamic quenching, the interaction of the fluorophore with the quencher is indirect. In contrast, in the case of static quenching, a ground state complex formation exists between the fluorophore and quencher [30]. Therefore, the FQM can be sorted out based on their temperature dependence. Furthermore, in the case of static quenching, Ksv values are inversely proportional to temperature, whereas in dynamic Ksv, the values are directly proportional to temperature. Therefore, the FQM of the HSA-QTP system was evaluated by recording the fluorescence spectra of HSA-QTP at different temperatures (295, 300, and 305 K), and the fluorescence quenching data of the HSA-QTP system was analyzed using the Stern-Volmer equation [30]:

$$\frac{\mathbf{F\_0}}{\mathbf{F}} = \mathbf{1} + \mathbf{K\_{sv}}[\mathbf{Q}] \tag{1}$$

where F<sup>0</sup> and F represent the steady-state fluorescence of HSA and the HSA-QTP complex, respectively. [Q] represents the quencher concentration (QTP), and Ksv represents the Stern-Volmer constant. The Ksv plot for the HSA-QTP system obtained at various temperatures (295, 300, and 305 K) is given in Figure 3B. It is found that the Ksv values for the HSA-QTP system decreased with a temperature rise, confirming the static quenching mechanism for the HSA-QTP system (Table 1). In addition, the fluorescence mechanism (quenching) was also analyzed according to the bimolecular rate constant values using the equation:

$$\mathbf{k\_{q}} = \mathbf{K\_{sv}} / \tau\_{0} \tag{2}$$

where k<sup>q</sup> is the bimolecular rate constant, and τ<sup>0</sup> is the average lifetime of the protein in the absence of the quencher and is valued at 10−<sup>8</sup> for biopolymers [35]. The calculated bimolecular quenching rate constant value for the HSA-QTP system is presented in Table 1. The k<sup>q</sup> values were found to be higher than the value of the scattering collision constant (2 <sup>×</sup> <sup>10</sup><sup>10</sup> <sup>M</sup>−<sup>1</sup> s −1 ), which again suggests the involvement of a static quenching mechanism between the HSA-QTP system [36].

Volmer equation [30]:


**Table 1.** The values of the Stern-Volmer constant and quenching rate constant for the QTP-HSA system.

2.2.2. Evaluation of the Binding Constants and the Number of Binding Sites in the HSA-QTP System

Intrinsic fluorescence data at different temperatures (295, 300, and 305 K) were used to determine the binding constant (Kb) and binding stoichiometry (n) of the HSA-QTP system by using the following equation [10,30]:

$$\log\frac{(\mathbf{F}\_0 - \mathbf{F})}{\mathbf{F}} = \log|\mathbf{K\_b} + \mathbf{n}\,\log[\mathbf{Q}]\,\tag{3}$$

where F<sup>0</sup> and F represent fluorescence intensities of HSA with or without the quencher (QTP), respectively. K<sup>b</sup> and n represent the binding constant and binding stoichiometry in the HSA-QTP system. The double log plot of log [(F<sup>0</sup> − F)/F] vs. log [Q] (Figure 3C) was used for the determination of the binding constant and binding stoichiometry. The values of K<sup>b</sup> and n were calculated from the intercept and slope of the plot, as shown in Figure 3C. As per Figure 3C, K<sup>b</sup> and n at different temperatures for the HSA-QTP system are presented in Table 2. A decrease in the binding constant was observed at higher temperatures for the HSA-QTP system. Further, the binding constants were ~10<sup>4</sup> , suggesting a moderate binding between HSA and QTP.

**Table 2.** The binding constant values and the number of binding sites for the interaction of QTP with HSA.


2.2.3. Determination of the Binding Forces between HSA and QTP-Thermodynamic Analysis

The primary binding intermolecular forces that are involved in the drug-protein interactions were estimated via thermodynamic parameters. The protein-drug interactions are held together by hydrophobic interactions, hydrogen bonds, electrostatic forces, and van der Waal interactions. Moreover, the sign and magnitude of the enthalpy (∆H<sup>0</sup> ) and entropy (∆S 0 ) change to determine the nature of binding forces in the drug-protein complex. For the hydrophobic interactions, the sign and magnitude must have a positive value for ∆H<sup>0</sup> and ∆S 0 . At the same time, in the case of van der Waals forces and hydrogen bonding, it must be negative for ∆H<sup>0</sup> and ∆S 0 [36,37]. Additionally, for the electrostatic interaction, ∆H<sup>0</sup> should be negative and ∆S <sup>0</sup> positive. The free energy (∆G<sup>0</sup> ) change of the HSA-QTP system can be determined by using the van 't Hoff equation and the thermodynamic equation given below:

$$
\ln \text{K}\_{\text{b}} = -\frac{\Delta \text{H}^{0}}{\text{RT}} + \frac{\Delta \text{S}^{0}}{\text{R}} \tag{4}
$$

$$
\Delta \mathbf{G}^0 = \Delta \mathbf{H}^0 - \mathbf{T} \Delta \mathbf{S}^0 \tag{5}
$$

where R represents the gas constant (8.314 J mol <sup>−</sup><sup>1</sup> K <sup>−</sup><sup>1</sup> ), T is the temperature in kelvins, and K<sup>b</sup> represents the binding constant at the studied different temperatures. ∆H<sup>0</sup> and ∆S 0 are obtained from the slope and intercept of the plot between lnK and 1/T (Figure 3D). The results of ∆G<sup>0</sup> , ∆H<sup>0</sup> , and ∆S <sup>0</sup> obtained from HSA-QTP interactions are summarized in Table 3. The positive values of ∆H<sup>0</sup> and ∆S 0 for the HSA-QTP system suggest that hydrophobic interactions played a significant role in the binding process of QTP to HSA. Thus, the formation of the HSA-QTP complex was exothermic and spontaneous [38].


**Table 3.** Various thermodynamic parameters for QTP-HSA complex formation at various temperatures.

2.2.4. Synchronous Fluorescence Spectroscopy (SFS) Experiment

The synchronous fluorescence spectrometry helps to provide information about the local environment of proteins around W and Y residues upon interaction with ligands [30,39]. In this experiment, the fluorescence difference between excitation and emission wavelengths reflects the nature of the spectra. A difference of wavelength (∆λ) of 15 nm is characteristic for (Y), and 60 nm is typical of (W) residues. Therefore, any shift in the maximum emission wavelength reflects the local environment changes around aromatic amino acid residue (Y and W) [40]. The SFS emission spectra of the HSA-QTP complex are given in Figure 4A,B. It was clear from Figure 4 that the HSA fluorescence intensity of both (W and Y) regularly decreases with the addition of QTP. Further, no shift in the emission wavelength was observed for either of the spectra at ∆λ = 15 nm or 60 nm. The HSA-QTP interaction did not lead to any microenvironmental change in the protein molecule upon interaction. *Molecules* **2022**, *27*, x FOR PEER REVIEW 7 of 17

**Figure 4.** Synchronous fluorescence spectra at Δλ = 15 nm (**A**) and Δλ = 60 nm (**B**) of HSA (5 μM) in the absence and presence of increasing concentrations of QTP (0-35 μM). At Δλ = 15 nm (for Y), the excitation wavelength of HSA was fixed at 240 nm, and the emission range was 255–400 nm, whereas at Δλ = 60 nm (for W), the excitation wavelength was taken at 240 nm and the emission range was 300–400 nm. **Figure 4.** Synchronous fluorescence spectra at ∆λ = 15 nm (**A**) and ∆λ = 60 nm (**B**) of HSA (5 µM) in the absence and presence of increasing concentrations of QTP (0-35 µM). At ∆λ = 15 nm (for Y), the excitation wavelength of HSA was fixed at 240 nm, and the emission range was 255–400 nm, whereas at ∆λ = 60 nm (for W), the excitation wavelength was taken at 240 nm and the emission range was 300–400 nm.

### 2.2.5. Binding and Prediction of Site Markers in the HSA-QTP System 2.2.5. Binding and Prediction of Site Markers in the HSA-QTP System

A site marker displacement experiment was investigated to identify QTP binding site on HSA. In this experiment, here warfarin (WAR) for Sudlow's site I (subdomain IIA), ibuprofen (IBU) for Sudlow's Site II (subdomain IIIA), and hemin (HEM) for binding site III (subdomain IB) were used as HSA site marker probes; [10,26]. As a result, fluorescence spectra were recorded HSA-QTP system in the presence of site marker probes (0-30 μM). Moreover, the displacement percentage (I %) of QTP with the site markers is estimated by the following methods [40,41]: A site marker displacement experiment was investigated to identify QTP binding site on HSA. In this experiment, here warfarin (WAR) for Sudlow's site I (subdomain IIA), ibuprofen (IBU) for Sudlow's Site II (subdomain IIIA), and hemin (HEM) for binding site III (subdomain IB) were used as HSA site marker probes; [10,26]. As a result, fluorescence spectra were recorded HSA-QTP system in the presence of site marker probes (0–30 µM). Moreover, the displacement percentage (I%) of QTP with the site markers is estimated by the following methods [40,41]:

$$\text{I(\%)}=\frac{\text{F}\_2}{\text{F}\_1} \times 100\tag{6}$$

I(%) = F1 × 100 (6) F<sup>1</sup> and F<sup>2</sup> represent the fluorescence emission intensities of the HSA-QTP system in the absence and presence of different site markers, respectively. However, the percentage F<sup>1</sup> and F<sup>2</sup> represent the fluorescence emission intensities of the HSA-QTP system in the absence and presence of different site markers, respectively. However, the percentage of displacement values of the HSA-QTP complex against the different concentrations of site

of displacement values of the HSA-QTP complex against the different concentrations of site markers is shown in Figure 5. It is apparent from Figure 5 that the displacement per-

binding site of QTP is predicted to be in site III (subdomain IB) of HSA.

markers is shown in Figure 5. It is apparent from Figure 5 that the displacement percentage of QTP from HSA by hemin is appreciably higher than WAR and IBU. Thus, the binding site of QTP is predicted to be in site III (subdomain IB) of HSA. *Molecules* **2022**, *27*, x FOR PEER REVIEW 8 of 17

**Figure 5.** Effect of site probes on the fluorescence emission intensities of the HSA-QTP system. The experiments were carried out using three site probes (warfarin, ibuprofen, and hemin). (HSA = 5 **Figure 5.** Effect of site probes on the fluorescence emission intensities of the HSA-QTP system. The experiments were carried out using three site probes (warfarin, ibuprofen, and hemin). (HSA = 5 µM, QTP = 10 µM, C = 0-30 µM), λex = 295 nm, T = 295 K.
