In Vitro and In Silico Analysis of the Bindings between Legacy and Novel Per- and Polyfluoroalkyl Substances and Human Serum Albumin

Per- and polyfluoroalkyl substances (PFASs) are emerging contaminants of concern that can enter the human body through a variety of pathways and thereby cause harmful effects. Exposure of pregnant women to PFASs could even affect both the mother and the child. Human serum albumin (HSA) is considered to be the primary transport protein for a variety of substances in body fluids. It can be bound to different contaminants and might result in possible effects on human health. Yet, few studies are available on the binding affinity of legacy PFASs and their novel alternatives to HSA. In this study, the binding mechanisms of HSA to both legacy PFASs and their novel alternatives were investigated using fluorescence spectroscopy, together with further molecular docking. The results show that all the target PFASs were statically quenched against HSA with binding ratios of 1:1. The binding constants of long-chain PFASs and novel alternatives of perfluoroalkanesulfonic acids (PFSAs) were greater than 102, whereas those of short-chain PFASs alternatives and novel alternatives of perfluorocarboxylic acids (PFCAs) were less than 102. In general, the binding affinities of PFCAs on HSA were less than that of PFSAs, while the binding affinities of short-chain PFASs alternatives on HSA were smaller than those of long-chain PFASs and their novel alternatives. Therefore, bindings to HSA could be considered as an important influencing factor for the bioaccumulation of legacy and novel PFASs in the human body.


Introduction
Per-and polyfluoroalkyl substances (PFASs), which are a family of chemical substances containing one or more perfluoroalkyl moieties (-C n F 2n+1 -), have been used globally as highly effective surfactants and surface protectants for the past seventy years [1].Particularly, strong perfluoroalkyl moieties have unique properties, including exceptional resistance to environmental and biodegradation, thermal and chemical stability to oxidation, photolysis, and hydrolysis reactions, as well as hydrophobic and oleophobic properties [2].Long-chain perfluoroalkyl carboxylic acids (PFCAs) (containing seven or more perfluorinated carbons) and perfluoroalkanesulfonic acids (PFSAs) (containing six or more perfluorinated carbons) are ubiquitous around the globe and thus have received much attention [3,4].Two decades ago, perfluorooctane sulfonate (PFOS) was found for the first time in the blood of wild animals and even in human blood [5,6].Studies have shown that long-chain PFASs are potentially toxic and may cause reproductive and developmental defects, hepatotoxicity, neurotoxicity, and immunotoxicity [7,8].In 2009, PFOS, its salts, and its precursor perfluorooctane sulfonyl fluoride (PFOSF) were restricted globally [9].Furthermore, another two long-chain PFASs, perfluorooctanoic acid (PFOA) and perfluorohexane sulfonic acid (PFHxS), their salts, and related compounds were phased out from international productions in 2019 and 2022, respectively [10,11].
Human serum albumin (HSA) is a globular protein that is a single peptide chain consisting of 585 amino acid residues.HSA has 30 phenylalanine residues, 35 cysteine residues, 18 tyrosine residues, and one tryptophan residue.The N-terminal is an aspartic acid residue and the C-terminal is a leucine residue.A sulfhydryl group is present at position 34 of the single peptide chain.These amino acid residues play a very important role in maintaining the spatial structure of HSA [25].Smeltz et al. [26] evaluated the in vitro human plasma protein binding (PPB) of 71 PFASs through ultracentrifugation and liquid chromatography-mass spectrometry analysis.The results revealed that perfluoroalkanoyl chlorides and PFCAs with 6-10 carbons were the highest bound PFASs, with similar median values for alkyl, ether, and polyethylene PFCAs.Pan et al. [27] demonstrated that HSA binding to pollutants could affect the efficiency of placental transfer.Experiments have shown that umbilical cord serum albumin can promote placental transfer of PFASs, whereas maternal serum albumin can reduce the efficiency of transfer.Previous studies have shown that factors such as the carbon chain length, functional groups, and structure (linear and isomeric) of PFASs may affect the binding to HSA.Longer carbon chains could hinder the binding of PFASs to proteins.However, there were different conclusions on the effect of carbon chain length of PFASs on binding to the protein.Previous studies from Li et al. [28] have demonstrated through experiments that various PFASs can be bound to HSA and have found that PFASs have high binding potential with HSA.Alesio et al. [29] analyzed the binding of PFASs to bovine serum albumin (BSA) based upon fluorescence quenching and obtained the binding constants, which are related to the physicochemical properties of PFASs.Recently, MacManus-Spencer et al. [30] compared three experimental methods to detect the binding of PFCAs to serum albumin and found that although fluorescence spectroscopy is an indirect method, it can more comprehensively describe the properties of the interaction.Moreover, Chen et al. [31] also analyzed the binding of PFCAs to HSA using the fluorescence spectroscopy and the interaction forces.Yet, few studies employing the fluorescence spectroscopy are available on the bindings between novel PFASs alternatives and HSA so far.
To better explore the bindings between PFASs and HSAs, the bindings can be modeled using molecular docking techniques.Molecular docking is an effective method to study intermolecular forces, especially for biomolecular complexes, such as the forces between Toxics 2024, 12, 46 3 of 16 drugs and receptors.Molecular docking can be applied to acquire information about the conformation, binding site, and binding force of the binding between ligand and receptor [32].The employment of molecular docking techniques not only saves a lot of time, but also provides the binding parameters of the ligand and receptor more quickly and directly.One study used molecular docking to observe the binding of PFOS and HSA and obtained information on the binding energy as well as the binding site, showing the maximum number of ligands that can bind to HSA was 9 for PFOA and 11 for PFOS, and that both of them had the highest binding free energy near the Trp214 binding site [33].Another researcher utilized molecular docking to explore the mechanism of PFASs transfer through the human placenta.It was shown that the binding affinity of PFASs to HSA increased with growing carbon chain length [34].So far, most studies have focused on revealing the binding mechanism between legacy PFASs and HSA, yet little information is available about the binding mechanism between novel PFASs alternatives and HSA.
In the present study, three legacy long-chain PFASs, including PFOS, PFOA, and PFHxS, three short-chain PFASs alternatives, including PFPrS, PFBS, and PFBA, together with four novel PFASs alternatives, 9Cl-PF3ONS, 11Cl-PF3OUdS, NaDONA, and HFPO-DA, were adopted as the target legacy and novel PFASs to achieve the following goals: (1) proving experimentally whether 10 PFASs would be bound with HSA and obtaining the information of binding sites and binding constants through the fluorescence spectroscopy; (2) deriving further information of binding sites and binding energy via the molecular docking technique; and (3) based upon all the obtained results, exploring the differences in the bindings of legacy and novel PFASs to HSA.

Experimental Methods for Three-Dimensional Fluorescence Spectroscopy
Briefly, 5 mL of HSA stock solution was mixed with 1 mL, 3 mL, and 5 mL of PFASs stock solution individually to obtain the concentration ratios of 5:1, 5:3, and 5:5, respectively, at room temperature, and then the mixtures were allowed to stand at the room temperature for 30 min.Then, 4 mL of each was taken, and the three-dimensional fluorescence spectra of the mixtures were measured by a fluorescence spectrophotometer (F-4700, Hitachi, Tokyo, Japan) to determine the optimal concentration for the instrumental analysis.Consequently, the following concentration ratios were selected by comparison.On one side, 5 mL of HSA stock solution was mixed with 1 mL of PFBS, PFHxS, PFOS, PFBA, PFOA, 9Cl-PF3ONS, and 11Cl-PF3OUdS stock solution to obtain a mixture with a concentration ratio of 5:1.On the other side, 5 mL of HSA stock solution was mixed with 3 mL of PFPrS, NaDONA, and HFPO-DA stock solutions to obtain a mixture with a concentration ratio of 5:3.The three-dimensional fluorescence spectra of the HSA stock solution were measured simultaneously.The excitation wavelength (λ ex ) ranged from 200-340 nm, the emission wavelength (λ em ) ranged from 270-460 nm, and the excitation and emission wavelength intervals were both 5 nm.

Molecular Docking Simulation Strategy
The crystal structure of HSA was taken from the Protein Structure Database (Protein Data Bank) with PDB code 4E99 [35].The structure of PFASs was drawn using Chemdraw 21.0.0.and optimized for the minimum energy to reach the lowest energy state of the chemical structure [36].Molecular docking simulations of PFASs binding with HSA were performed using Autodock Vina (v 1.1.2)[37].A grid box of 126-126-126 with a spacing of 0.581 Å was used in order to include all possible binding sites for each PFAS to HSA.The center grid box of x, y, and z was 0.861, 1.278, and −4.167, respectively.Each simulation generated 10 binding modes and the value of exhaustiveness is 10.Molecular docking results were visualized and analyzed using PyMOL 2.5.2.[38].

Data Analysis
The fluorescence quenching data were usually analyzed by Modified Stern-Volmer Equations ( 1) and ( 2) [39] F 0 F 0 refers to the luminescence intensity of HSA in the absence of PFASs, and F refers to the luminescence intensity of HSA in the presence of different amounts of PFASs.K ′ SV is the slope of the modified Stern-Volmer curve, which is the quenching constant of the interacting system.τ 0 refers to the average lifetime of the fluorescent substance in the absence of a fluorescent quenching agent.The average fluorescence lifetime of biomolecules is generally 10 −8 s.
When small molecules bind independently to a set of equivalent sites on a macromolecule, the equilibrium between free and bound molecules is given by the Lineweaver-Burk double logarithmic Equation (3) [40] lg K A denotes the binding constant of PFASs to HSA, and n denotes the number of apparent binding sites between them.

Three-Dimensional Fluorescence Spectroscopy Analysis
Three-dimensional fluorescence spectroscopy can intuitively reflect the protein fluorescence intensity and its conformational change information, which can be used for the analysis of HSA conformation and microenvironment [41].Two distinct fluorescence peaks (peak 1, λ em = 235 nm, λ ex = 325 nm, I 1 = 362.3and peak 2, λ em = 280 nm, λ ex = 335 nm, I 2 = 1803), together with a first-order Rayleigh scattering peak (peak 3, λ ex = λ em = 290 nm, I 3 = 1058) can be seen in the three-dimensional fluorescence spectrogram of HSA (Figure 1a).Among them, peak 1 reflects information such as the structure of the protein polypeptide chain, while peak 2 reflects the spectral behavior of the tryptophan and tyrosine residues of HSA [42,43].The three-dimensional fluorescence spectra of long-chain PFASs before and after binding with HSA are shown in Figure 1b-d.residues of HSA [42,43].The three-dimensional fluorescence spectra of long-chain PFASs before and after binding with HSA are shown in Figure 1b-d.Similarly, the fluorescence spectra of short-chain PFASs bound to HSA and fluorescence spectra of PFASs alternatives bound to HSA were measured as shown in Figures 2  and 3.However, the fluorescence peak intensities of PFPrS, NaDONA, and HFPO-DA did not change significantly.This might be because the binding constants of these three alternatives for long-chain PFASs with HSA were lower, so their concentration ratios needed to be increased to observe the changes.Therefore, the three-dimensional fluorescence spectra of HSA with a concentration ratio of 5:3 of these three PFASs were measured accordingly, and the changes could be clearly observed.Similarly, the fluorescence spectra of short-chain PFASs bound to HSA and fluorescence spectra of PFASs alternatives bound to HSA were measured as shown in Figures 2  and 3.However, the fluorescence peak intensities of PFPrS, NaDONA, and HFPO-DA did not change significantly.This might be because the binding constants of these three alternatives for long-chain PFASs with HSA were lower, so their concentration ratios needed to be increased to observe the changes.Therefore, the three-dimensional fluorescence spectra of HSA with a concentration ratio of 5:3 of these three PFASs were measured accordingly, and the changes could be clearly observed.residues of HSA [42,43].The three-dimensional fluorescence spectra of long-chain PFASs before and after binding with HSA are shown in Figure 1b-d.Similarly, the fluorescence spectra of short-chain PFASs bound to HSA and fluorescence spectra of PFASs alternatives bound to HSA were measured as shown in Figures 2  and 3.However, the fluorescence peak intensities of PFPrS, NaDONA, and HFPO-DA did not change significantly.This might be because the binding constants of these three alternatives for long-chain PFASs with HSA were lower, so their concentration ratios needed to be increased to observe the changes.Therefore, the three-dimensional fluorescence spectra of HSA with a concentration ratio of 5:3 of these three PFASs were measured accordingly, and the changes could be clearly observed.

Two-Dimensional Fluorescence Spectral Analysis
The interaction between organic small molecules and proteins can lead to the protein fluorescence quenching phenomenon, which can be divided into two forms, i.e., dynamic quenching and static quenching.Of these, static quenching refers to the phenomenon wherein organic small molecules and proteins combine with each other to form complexes with the help of intermolecular forces, which leads to the phenomenon of weakening of the fluorescence intensity of proteins [44].Through the calculation of the fluorescence quenching constant, binding constant, and thermodynamic constant, a preliminary judgment can be made on the mutual binding site and the form of force between small molecules and proteins [45].

Two-Dimensional Fluorescence Spectral Analysis
The interaction between organic small molecules and proteins can lead to the protein fluorescence quenching phenomenon, which can be divided into two forms, i.e., dynamic quenching and static quenching.Of these, static quenching refers to the phenomenon wherein organic small molecules and proteins combine with each other to form complexes with the help of intermolecular forces, which leads to the phenomenon of weakening of the fluorescence intensity of proteins [44].Through the calculation of the fluorescence quenching constant, binding constant, and thermodynamic constant, a preliminary judgment can be made on the mutual binding site and the form of force between small molecules and proteins [45].
In order to clarify the mechanism of endogenous fluorescence quenching of HSA by PFASs, the effects of different concentrations of PFASs on the fluorescence spectrum of HSA were investigated.From A to H, the concentration ratios of HSA to PFASs were 5:0, 5:1, 5:5, 5:10, 5:15, 5:20, 5:30, and 0:5, respectively.Under 275 nm light excitation, the intensity of HSA endogenous fluorescence gradually decreased with the increasing concentration of PFASs.Although there was no obvious change in the peak shape, the fluorescence emission peak was blue-shifted from 335 nm to 310 nm.This indicated that the target PFASs interacted with HSA.
The results of HSA combined with long-chain PFASs, short-chain PFASs, and novel PFASs alternatives are shown in Figures 4-6, respectively.intensity of HSA endogenous fluorescence gradually decreased with the increasing concentration of PFASs.Although there was no obvious change in the peak shape, the fluorescence emission peak was blue-shifted from 335 nm to 310 nm.This indicated that the target PFASs interacted with HSA.
The results of HSA combined with long-chain PFASs, short-chain PFASs, and novel PFASs alternatives are shown in Figures 4, 5, and 6, respectively.intensity of HSA endogenous fluorescence gradually decreased with the increasing concentration of PFASs.Although there was no obvious change in the peak shape, the fluorescence emission peak was blue-shifted from 335 nm to 310 nm.This indicated that the target PFASs interacted with HSA.
The results of HSA combined with long-chain PFASs, short-chain PFASs, and novel PFASs alternatives are shown in Figures 4, 5, and 6, respectively.intensity of HSA endogenous fluorescence gradually decreased with the increasing concentration of PFASs.Although there was no obvious change in the peak shape, the fluorescence emission peak was blue-shifted from 335 nm to 310 nm.This indicated that the target PFASs interacted with HSA.
The results of HSA combined with long-chain PFASs, short-chain PFASs, and novel PFASs alternatives are shown in Figures 4, 5, and 6, respectively.

Calculation of Fluorescence Quenching Rate Constants
The fluorescence quenching behavior of HSA was calculated using Equations ( 1) and ( 2) [39].The modified Stern-Volmer curves with the inverse concentration of PFASs as the horizontal coordinate and F 0 (F 0 −F) as the vertical coordinate are shown in Figures 7-9.The fitted equations, R 2 , and fluorescence quenching rate constant K q were shown in Tables 1-3.
The modified Stern-Volmer curves with the inverse concentration of PFASs as the horizontal coordinate and as the vertical coordinate are shown in Figures 7-9.The fitted equations, R 2 , and fluorescence quenching rate constant Kq were shown in Tables 1-3.The order of magnitude of Kq was calculated to be between 10 10 and 10 11 in the present study.The maximum scatter collision quenching constant, Kq, of various quenchers with the biopolymer was reported elsewhere to be 2.0 × 10 10 L/mol/s [46].Therefore, the fluorescence quenching of PFASs on HSA was not formed by the mutual collision of PFASs on HSA to form a dynamic quenching process, but the formation of a complex might re-    The order of magnitude of K q was calculated to be between 10 10 and 10 11 in the present study.The maximum scatter collision quenching constant, K q , of various quenchers with the biopolymer was reported elsewhere to be 2.0 × 10 10 L/mol/s [46].Therefore, the fluorescence quenching of PFASs on HSA was not formed by the mutual collision of PFASs on HSA to form a dynamic quenching process, but the formation of a complex might result in a static quenching process [47,48].

Calculation of Binding Sites and Binding Constants
Based upon the results of PFASs binding on HSA as a static quenching process, the binding between the two conforms to the expression formula for the binding of fluorescent substances and quenching agents: Lineweaver-Burk double logarithmic Equation (3) [40].The binding constants and apparent binding sites were calculated for the system of interaction between PFASs and HSA.
The curves obtained are shown in Figures 10-12.The fitting equations, R 2 , the number of binding sites n, and the binding constant K A were shown in Tables 4-6.The n values of the fitting equations for the binding of HSA to the novel PFASs alternatives were all close to 1, which indicated that one HSA molecule could bind approximately one molecule of the novel PFASs alternatives, and quantitative analyses of the ratio of the two bindings are of great importance for the determination of the forces acting between them [49,50].
According to the results mentioned above, it was demonstrated that the binding constants of PFSAs were greater than those of PFCAs.The binding constants increased with increasing carbon chain length.Among the four novel PFASs alternatives, the binding constants of the sulfonic acid substitutes were higher than those of the carboxylic acid alternatives.9Cl-PF3ONS and 11Cl-PF3OUdS, as PFOS alternatives, showed that their binding constants with HSA were less than those of PFOS with HSA.Similarly, the bind-  The n values of the fitting equations for the binding of HSA to the novel PFASs alternatives were all close to 1, which indicated that one HSA molecule could bind approximately one molecule of the novel PFASs alternatives, and quantitative analyses of the ratio of the two bindings are of great importance for the determination of the forces acting between them [49,50].
According to the results mentioned above, it was demonstrated that the binding constants of PFSAs were greater than those of PFCAs.The binding constants increased with increasing carbon chain length.Among the four novel PFASs alternatives, the binding constants of the sulfonic acid substitutes were higher than those of the carboxylic acid alternatives.9Cl-PF3ONS and 11Cl-PF3OUdS, as PFOS alternatives, showed that their binding constants with HSA were less than those of PFOS with HSA.Similarly, the binding constants of NaDONA and HFPO-DA, as PFOA substitutes, were significantly smaller than the binding constants of PFOA to HSA.It was suggested that the binding constants of novel PFASs alternatives to HSA were lower than those of legacy PFASs.

Molecular Docking
Some previous studies have used molecular docking simulation techniques to investigate the combination of different PFASs and HSA.Delva-Wiley et al. [51] used molecular docking technology to determine four different binding sites between GenX and HSA.Salvalaglio et al. [33] calculated the number of binding sites between PFOA/PFOS and HSA using the molecular docking techniques.Li et al. [28] calculated the binding affinities of different PFASs and HSA using molecular docking techniques, which can effectively separate PFASs bound to HSA.In this study, molecular docking simulations of HSA with PFASs molecules were performed using Autodock Vina.The conformation with the lowest binding energy for each target PFAS were selected for further analysis.
As shown in Figures 13 and 14, green color represented carbon atoms, cyan color represented fluorine atoms, red color represented oxygen atoms, yellow color represented sulfur atoms, blue represented chlorine atoms, pink color represented amino acid residues, and the yellow dashed line represented hydrogen bonds.Negative figures for the binding energy indicated that the bindings of HSA and PFASs were an exothermic reaction that can occur spontaneously.The larger the absolute value of binding energy, the more energy is released during the synthesis of the complex.Accordingly, the more energy is released, the lower the system energy is retained, and the formed complex is hence more stable [52].As demonstrated in Table 7, the binding energy between long-chain PFASs and HSA is lower than that of short-chain PFASs alternatives.When the carbon chains are the same, the binding energy of PFSAs is lower than that of PFCAs.As the carbon chain increases, the binding energy decreases.For novel PFASs alternatives, the binding energy of PFOA is lower than its alternatives of NaDONA and HFPO-DA.Similarly, the binding energy of PFOS is lower than its alternatives of 9Cl-PF3ONS and 11Cl-PF3OUdS.Furthermore, the binding energy of short-chain PFASs alternatives is lower than that of novel PFASs alternatives.It is revealed that the complexes between three long-chain PFASs and HSA are more stable than their short-chain and novel alternatives.carbon chain increases, the binding energy decreases.For novel PFASs alternatives, the binding energy of PFOA is lower than its alternatives of NaDONA and HFPO-DA.Similarly, the binding energy of PFOS is lower than its alternatives of 9Cl-PF3ONS and 11Cl-PF3OUdS.Furthermore, the binding energy of short-chain PFASs alternatives is lower than that of novel PFASs alternatives.It is revealed that the complexes between three longchain PFASs and HSA are more stable than their short-chain and novel alternatives.Combining the molecular docking results with the experimental results above, it can be summarized that the binding ability of PFSAs to HSA is usually higher than that of PFCAs.Compared to long-chain PFASs, the binding of short-chain PFASs alternatives to HSA is less stable, while the binding ability of novel PFASs alternatives to HSA is lower than that of long-chain PFASs, but higher than that of short-chain PFASs alternatives, which was also described in a previous study [53].The lower binding affinity of PFASs alternatives shows that they are less likely to be accumulated in the human body.Therefore, it is also suggested that short-chain and novel PFASs alternatives might tend to transport from maternal sera to cord sera through the placenta, due to their lower binding affinity to the placental HSA.Consistently, our recent study based upon the comprehensive analysis of 21 legacy and 49 novel PFASs in 50 paired maternal-infant serum samples around Fuxin fluorochemical facilities determined that legacy PFASs tended to exist in the maternal sera, whereas novel PFASs alternatives tended to exist in the sera of infants [19].

Conclusions
In summary, the bindings of HSA with legacy and novel PFASs alternatives were initially observed using a three-dimensional fluorescence spectrogram, and the results proved the bindings of HSA and all the target PFASs.The fluorescence quenching mechanism of HSA was then investigated using a two-dimensional fluorescence spectrogram.With the increasing concentration of PFASs, the fluorescence intensity of HSA was gradually decreased and the fluorescence emission peak was blue-shifted from 335 nm to 310 nm, which indicated that the fluorescence quenching of HSA occurred.Moreover, calculations using modified Stern-Volmer equations showed that the quenching constants were greater than 2.0 × 10 10 L/mol/s, representing that the quenching mechanism of HSA was static quenching.The binding constants as well as the numbers of binding sites were also calculated using the Lineweaver-Burk double logarithmic equation, showing that the binding ratios of all the PFASs to HSA were 1:1.Except for PFPrS, the binding constants of PFSAs and their alternatives were all greater than 10 2 , and the binding constant increased with the growth of the carbon chain.Except for PFOA, the binding constants of PFCAs and their alternatives were all less than 10 2 , and the binding constant declined as the carbon chain decreased.Notably, the binding constants of novel PFASs alternatives were smaller than those of legacy PFASs.Furthermore, the molecular docking technique was used to simulate the bindings between HSA and each target PFAS, revealing that the binding energies between legacy long-chain PFASs and HSA were usually lower than those of short-chain and novel PFASs alternatives, and the binding energies between HSA and PFSAs were usually smaller than those of HSA and PFCAs.It was demonstrated that, compared to legacy long-chain PFASs, short-chain PFASs and novel PFASs alternatives could be less likely to be accumulated in the human body and have higher mobility because of their

Figure 9 .
Figure 9. Modified Stern-Volmer equations diagram of HSA combined with novel PFASs alternatives.

Figure 9 .
Figure 9. Modified Stern-Volmer equations diagram of HSA combined with novel PFASs alternatives.

Figure 13 .
Figure 13.A zoomed-out view of the binding pocket of HSA and long-chain PFASs, short-chain PFASs alternatives, and novel PFASs alternatives.

Figure 13 .
Figure 13.A zoomed-out view of the binding pocket of HSA and long-chain PFASs, short-chain PFASs alternatives, and novel PFASs alternatives.

Table 1 .
The fitted equations and fluorescence quenching rate constants of long-chain PFASs.

Table 1 .
The fitted equations and fluorescence quenching rate constants of long-chain PFASs.

Table 2 .
The fitted equations and fluorescence quenching rate constants of short-chain PFASs.

Table 3 .
The fitted equations and fluorescence quenching rate constants of novel PFASs alternatives.

Table 1 .
The fitted equations and fluorescence quenching rate constants of long-chain PFASs.

Table 2 .
The fitted equations and fluorescence quenching rate constants of short-chain PFASs.

Table 3 .
The fitted equations and fluorescence quenching rate constants of novel PFASs alternatives.

Table 5 .
The fitted equations, binding constants, and the numbers of binding sites of short-chain PFASs.

Constant KA The Number of Binding Sites n Figure
11. Lineweaver-Burk double logarithmic equation diagram of HSA combined with shortchain PFASs.

Table 6 .
The fitted equations, binding constants, and the numbers of binding sites of novel PFASs alternatives.

Table 4 .
The fitted equations, binding constants, and the numbers of binding sites of long-chain PFASs.

Table 5 .
The fitted equations, binding constants, and the numbers of binding sites of shortchain PFASs.

Table 6 .
The fitted equations, binding constants, and the numbers of binding sites of novel PFASs alternatives.

Table 7 .
Simulated binding energies and binding residues of HSA to three types of PFASs via molecular docking.
Figure 14.Molecular docking diagram of HSA and long-chain PFASs, short-chain PFASs alternatives, and novel PFASs alternatives.

Table 7 .
Simulated binding energies and binding residues of HSA to three types of PFASs via molecular docking.