The interaction mechanism between fludarabine and human serum albumin researched by comprehensive spectroscopic methods and molecular docking technique

Journal Pre-proof The interaction mechanism between fludarabine and human serum albumin researched by comprehensive spectroscopic methods and molecular docking technique

XiaoLe Han, Hao Hao, QingYu Li, ChenYin Liu, JiaWen Lei, Fan Yu, Ke Chen, Yi Liu, Tao Huang PII:

S1386-1425(20)30148-7

DOI:

https://doi.org/10.1016/j.saa.2020.118170

Reference:

SAA 118170

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date:

15 December 2019

Revised date:

3 February 2020

Accepted date:

16 February 2020

Please cite this article as: X. Han, H. Hao, Q. Li, et al., The interaction mechanism between fludarabine and human serum albumin researched by comprehensive spectroscopic methods and molecular docking technique, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2020), https://doi.org/10.1016/j.saa.2020.118170

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier.

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The Interaction Mechanism Between Fludarabine And Human Serum Albumin Researched By Comprehensive Spectroscopic Methods And Molecular Docking Technique

Key Laboratory of Catalysis and Energy Materials Chemistry of Ministry of

Education

&

Hubei Key Laboratory of Catalysis and Materials Science,

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XiaoLe Han,a,*,# Hao Hao,a,# QingYu Li,a ChenYin Liu,a JiaWen Lei,a Fan Yu,a Ke Chen,a Yi Liu,b Tao Huanga,*

South-Central University for Nationalities, Wuhan 430074, China. State Key Laboratory of Virology & Key laboratory of Analytical Chemistry for

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Biology and Medicine (MOE), College of Chemistry and Molecular Sciences, Wuhan

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University, Wuhan 430072, P. R. China.

________________________________ *corresponding authors. E-mail address: [email protected] (XiaoLe Han) [email protected] (Tao Huang） 1

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Abstract Fludarabine (Flu) is widely used to treat B-cell chronic lymphocytic leukemia. HSA is of the essence to human, especially in blood circulation system. The interaction mechanism between Flu and HSA was studied by comprehensive spectroscopic methods and molecular docking technique. UV-vis and FL spectrum results indicated that Flu bond with HSA, and there was a new complex produced at the binding site Ⅰ

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in subdomain ⅡA. Association constants at 298 K were 1.637×104 M−1 and 1.552×104

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M−1 at 310 K, respectively. The negative enthalpy (ΔH) and positive entropy (ΔS)

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values for the interaction revealed that the binding behavior was driven by

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hydrophobic forces and hydrogen bonds. The results obtained from UV, RLS spectra, 3D fluorescence and CD spectrum illustrated that Flu could change the secondary

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interaction is -11.15 kcal/mol.

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structure of HSA. According to molecule docking result, the binding energy of

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Keywords: Circular dichroism·docking·Fludarabine·Fluorescence spectroscopy·HSA

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Journal Pre-proof 1. Introduction Fludarabine (Flu) is a kind of purine analog, which is commonly considered to be an efficient agent for Chronic Lymphocytic Leukemia (CLL) and small lymphocytic lymphoma (SLL).[1] It is also used as the conditioning agent with other drug in nonmyeloablative allogeneic transplantation.[2-5] Before entering the cell, Flu is phosphorylated through intracellular kinases to release its cytotoxic activity.[6] In cancer cell molecules, Flu is dephosphorylated and forms active materials. Then it

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interferes with DNA synthesis and inhibits the chain elongation by arresting the

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duplication of DNA, or stopping RNA translation.[7-9] Avramis et al. found that Flu

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could kill the murine leukemic cells indiscriminately, hinder the repairation and the

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proliferation of normal cells in healthy leukocytes.[10,11] Moreover, Lichtman et al. reported most of active metabolite of Flu could be excreted by kidney. So the usage of

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patients.[12]

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Flu should be carefully, especially for those sick person with renal failure and senile

As the most pivotal carrier of blood plasma, HSA affects the pharmacokinetics of

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plentiful drugs and other bioactive agents. It almost takes up the half of blood, and maintains the serum osmotic pressure. HSA has three hydrophobic cavities in the subdomain IB, IIA, and IIIA, which has the excellent capability to bind with kinds of ligands.[13-22] Due to the unique features, albumin are the specific carrier to deliver targeted drug for research in oncology. However, if the binding affinity is too strong, drugs are metabolized more slowly in the body, which may cause biotoxicity.[23] Actually, there are many methods to determine the binding mechanism effectively. The most common methods are fluorescence spectroscopy and UV-vis absorbance spectroscopy due to convenience and having no special requirements for samples, which are the classic way to study the interactions between biomacromolecules and

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Journal Pre-proof small molecules[24]. Especially, the amino acid residues of protein have specific absorption peak and the fluorescence intensity of tryptophans is obvious and strong at λex = 280 nm[25,26]. In addition, time-resolved fluorescence spectroscopy could judge the mode of binding between small molecules and biomacromolecules[27,28]. Meanwhile, Fourier transform infrared spectroscopy could detect the change of proteins conformation, but it is difficult to obtain whole information of conformation change[29]. However, circular dichroism spectroscopy could obtain more detailed

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information of conformation change, such as the contents of α-helixes and β-sheets[30].

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Isothermal titration calorimetry (ITC) is a technique to detect the heating effect during

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the interaction process directly. Whereas, the samples for ITC must be with high

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requirements, such as samples’ solvent should be consistent as fully as possible, the concentration of drug solution should be high and binding affinity should be strong [31].

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Molecular docking technique is an advanced way to study the conformation changes

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of ligand and receptor molecules during the binding process by determining the fairly correct position and orientation of the ligand and receptor. In this paper, we employ

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the UV–vis absorption spectroscopy, resonance light-scattering spectra (RLS), fluorescence spectroscopy, time-resolved fluorescence spectroscopy, synchronous fluorescence, three-dimensional fluorescence, CD spectroscopy and molecular modeling technique to estimate the interaction mechanism between Flu and HSA, determine the mode of quenching mechanism and obtain thermodynamic information of interaction process. We also try to further determine the effects of Flu on the secondary structure of HSA. The investigation of interaction between HSA and Flu is obscured as yet. Therefore, the information of the binding behaviors between them will contribute the development of high-efficiency anticancer drug based on Flu. 2. Experimental

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Journal Pre-proof 2.1. Apparatus A UNICO 4802 UV-vis double-beam spectrophotometer was used to measure absorption spectra of HSA and HSA-Flu system. The absorption spectra were measured with a 1 cm quartz cell. The wavelength of the spectra was measured at a range of 200 to 500 nm. Fluorescence analyses were measured on an LS-55 fluorophotometer (PerkinElmer, USA) in the ratio mode with temperature maintained by circulating

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bath. The excitation wavelengths of HSA was 280 nm, and the emission wavelengths

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of HSA was 343 nm. Each spectrum was the average of three scans. Titrations were

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performed by trace syringes (5μL). Site competition spectra was obtained at the same

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condition of quenching spectra. Synchronous fluorescence spectra were obtained at a fixed excitation wavelength (Δλ = 15 nm and 60 nm). RLS spectra was obtained when

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the excitation wavelength equals to the emission wavelength. 3D spectra of both HSA

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and HSA-Flu system was obtained at excitation wavelength at 200 nm. The concentration of HSA was kept at 2 μM, and Flu of different concentrations were

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added into the solution.

The time-resolved emission measurements were measured on a FLS1000 spectroscopy instrument (Edinburgh Instruments, UK), the internal fluorescence of TRP214 in HSA and HSA-Flu system were measured at 300 nm to 500 nm, and with excitation at 280 nm. The concentrations of HSA and Flu were kept at 2μM. CD measurements were performed on a circular dichroism photomultiplier (Applied Photophysics Limited, UK) at 25 °C, which is equipped with quartz cells (path length is equal to 0.1 cm) . We set the scanning speed at 200 nm min−1. The CD spectra of HSA and the system of HSA-Flu were recorded in the range of 260-190 nm. Appropriate buffer solutions, measured under the same experimental conditions, were

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Journal Pre-proof taken as blanks and subtracted from the sample spectra. Each spectrum was the average of three scans. 2.2. Materials Human serum albumin (HSA), Fludarabine (Flu), dimethylsulfoxide (DMSO), ibuprofen and warfarin were obtained from Sigma-Aldrich. Sodium chloride (NaCl), potassium chloride (KCl), potassium phosphate dibasic (K2HPO4·12H2O), and potassium dihydrogen phosphate (KH2PO4) were purchased from Sinopharm

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Chemical Reagent Co. (China). All chemicals used in this study were of analytical

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reagent grade. All aqueous solutions were prepared with ultrapure water. All

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chemicals were commercially available and used without further purification.

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All the experiments were repeated three times. 3. Results and discussion

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3.1. The binding analysis of Flu and HSA

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3.1.1. The influence of Flu on the absorption of HSA UV-vis absorption measurement is a sensitive method to explore the structural

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changes and to know the complex formations.[25] Figure 1 (a) is the UV absorption spectrum of Flu in PBS only. It showed that the absorption of the Flu at 265 nm enhanced with the increasing concentration. The absorption of HSA titrated by Flu is shown in Figure 1 (b). It revealed that the UV absorption of HSA at 280 nm enhanced with increasing concentration of Flu and shifted from 280 nm to 270 nm. According to the Figure 2, the absorption spectrum by subtracting the absorption spectrum of Flu from that of HSA-Flu at the same concentration could be not overlapped within experimental error. Therefore, we conclude that the binding behavior between HSA and Flu is forming the complex. 3.1.2. The influence of Flu on the Resonance light-scattering spectra of HSA

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Journal Pre-proof RLS assay is a quick and convenient method to detect the binding behavior between biomacromolecules and small molecules. When the drug and protein interact, the vibrative electrons of protein absorb light strongly and scatter again.[26] From Figure 3 (a), we can see the scattering of electron of HSA strengthen regularly in the presence of Flu. In the other word, Flu can bind to HSA and form a new compound indeed. 3.1.3. The determination of the binding mechanism by Fluorescence data

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Fluorescence quenching is an phenomenon, which means the fluorescence

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intensity of a material decreases.[27] Quenching caused by molecular interactions,

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including excited-state reactions, molecular rearrangements, energy transfer,

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ground-state complex formation, and collisional quenching.[30] The quenching mechanism is classified as the static quenching and dynamic quenching mechanism.[31]

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In static quenching mechanism, the quencher and acceptor interact and generate

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ground-state complex. Whereas, the quencher and acceptor collisional encounters in dynamic quenching mechanism. Therefore, increasing the temperature results in

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thermal motion of system making diffusion coefficients bigger. On the contrary in static quenching mechanism, increasing temperature usually results in the dissociation of weakly bound complexes, and decreasing static quenching coefficients. These two mechanisms can be distinguished by different dependences on temperatures.[22] In this study, the mechanism was determined by examining the temperature-dependent fluorescence quenching behavior.[19,23] We studied the binding behavior of HSA-Flu system at the different temperatures. When the concentration of HSA was stabilized at 2×10-6 mol/L, and the concentration of Flu varied from 0 to 7×10-6 mol/L. The fluorescence intensity of HSA decreased regularly with the increasing of Flu concentration. By this situation we speculated that the interaction mechanism initially,

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Journal Pre-proof which is static quenching mechanism. According to the static quenching mechanism, the fluorescence quenching data are usually analyzed by the Stern-Volmer equation:

F0 (1)  1  K SV [Q] F Where, F0 and F are the steady-state fluorescence intensities in the absence and in the presence of quencher (Flu), respectively, KSV is the Stern-Volmer quenching constant, and [Q] is the concentration of quencher. The Stern-Volmer plot of HSA

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tryptophan residues fluorescence by Flu at 298 K, 301 K and 310 K is shown in Figure 3 (b).

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By using equation (1), the Stern-Volmer quenching constants KSV of HSA

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tryptophan residues fluorescence in presence of Flu at different temperatures (298,

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301, and 310 K) were obtained. The results are shown in Table 1，which indicated that

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the probable quenching mechanism between HSA and Flu is a static quenching procedure due to the KSV decreased with the temperature rising. As well as the value

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of KSV is four orders of magnitude, much less than the maximum diffusion collision quenching constant (2.0×1010 M-1s-1).[27] Therefore we consider that the interaction

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between HSA and Flu is due to ground complex, which is responding to the result of UV-vis absorption assay.

Therefore, the quenching data were analyzed according to the modified Stern-Volmer equation:[32] F0 F0 1 1    F F0 - F f a K a [Q] f a

(2)

In the present case, F0 and F are respectively the fluorescence intensity of HSA in the absence and presence of Flu; fa represents the mole fraction of solvent-accessible fluorophore; Ka is the effective quenching constant for the accessible fluorophore, analogous to the associative binding constant for the

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Journal Pre-proof quencher-acceptor.[33] As is shown in Figure 4, the binding constants are listed in Table 2. At temperatures ranging from 25 ℃ to 37 ℃, some of neutrally or (and) negatively charged exogenous substances bind to serum albumin with association constants between 103 and 105 mol/L,[16,21,25] which are similar to the (order of magnitude of) association constants showed in Table 2 in the order of magnitude. The experimental details of three times proved in Table S1 and Table S2 (in supporting information).

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The fluorescence transient decay curves of HSA and HSA-Flu system were

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shown in Figure 5. According to the calculation results, the average lifetimes of HSA

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and HSA-Flu system are 5.20 ns and 5.16 ns, respectively. The difference of HSA

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lifetime is little in presence of Flu. In dynamic quenching, excited molecules collide and their fluorescence life is reduced. Whereas, in static quenching, new complexes

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are formed between molecules, which have less effect on the fluorescence life of

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proteins. Combining fluorescence data with UV data, we further confirmed that HSA bound with Flu at ground state, formed a new complex and the interaction mechanism

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was static quenching mechanism.

3.1.4. Analysis of binding equilibrium and binding sites For static quenching, the fluorescence intensity is used to calculate the apparent binding constant (Kb) and the number of binding sites (n) using equation (3) when small molecules independently interact to a set of equivalent sites on a macromolecule:[34]

log

F0 - F  log Kb  n log[ Q] F The dependence of log

(3)

F0 - F on the value of log [Q] is linear with slope equal F

to the value of n and the value log Kb is fixed on the ordinate. The data around body 9

Journal Pre-proof temperature (310 K) were handled in this way and obtained the equation

log

F0 - F  3.744  0.976 log[ Q] for HSA. Therefore, the value of n was calculated F

to be 0.976, which indicated Flu has bond with HSA at approximately one binding site. Generally, there are two basic binding sites for protein. They are site I and site II, which usually marked by warfarin and ibuprofen, respectively.[35] We continued to determine the binding sites of Flu to HSA. The system of HSA only, HSA-warfarin,

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HSA-ibuprofen were titrated by Flu with the same concentration, respectively. The

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fluorescence data was analysed by equation (1) as in Figure 6. From Table 3, the

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results revealed that the KSV of HSA-ibuprofen is close to HSA only, but dramatically different of HSA-Warfarin. It indicated that only Warfarin competed with Flu for the

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site I in the subdomain IIA of HSA. That is to say, the binding site of Flu to HSA

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should be site I.

3.2. Thermodynamic parameters and interaction forces

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Generally, hydrophobic interactions, electrostatic attractions and repulsions, van

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der Waals interactions and hydrogen-bonding forces are the major interaction forces between a ligand and protein.[36,37] For confirming the nature of the interactions between Flu and HSA, the thermodynamic parameters are calculated according to the van’t Hoff equation:[38]

ln K a  -

H S  RT R

(4)

where R is the gas constant, ΔH is the enthalpy change and ΔS is the entropy change. And the free energy change (ΔG) can be calculated by the following equation :[38]

G  H - TS

(5)

A plot of ln Ka versus 1/T (inset in Figure 4) enables the determination of ΔH and 10

Journal Pre-proof ΔS for the binding at different temperatures (Table 2).[37] The negative enthalpy (ΔH) and positive entropy (ΔS) values for the interaction between Flu and HSA indicated that the electrostatic interactions, hydrophobic forces and hydrogen bonds may played major roles in the binding process.[36] But in our study, HSA was negative because the isoelectric point (4.7) of HSA was less than pH (7.4). Besides, Flu was phosphorylated easily. Therefore, electrostatic interactions did not work in this interaction procedure. Additionally, the negative free energy change (ΔG) indicated

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that the binding process is spontaneous.

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3.3. Conformational change

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The 3D fluorescence spectra for free HSA and HSA-Flu system shown in Figure

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6. There are two “humps” in the three-dimensional fluorescence spectra for HSA and HSA-Flu system, in which the peaks are marked peak 1 and peak 2.[39] The peak 1

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reflected the intrinsic fluorescence characteristics of Trp and Tyr residues of HSA,

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involving π → π* transition which is related to the changes in tertiary structure. The peak 2 symbolized the fluorescence characteristics of the polypeptide backbone

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structure of HSA due to the π→π* transition which is related to the changes in the secondary structure.[40,41] According the spectra, as is known that both of two fluorescence peaks of HSA have been quenched with the ratio of 14.6 % and 45.0 %, respectively. The results implied that Flu has formed a complex with HSA at ground state, thereafter changed its conformation. And the maximum excitation and emission wavelengths are 280.0 and 342.0 nm for peak 1 and 280.0 and 340.5 nm for peak 2. By contrast, the three-dimensional fluorescence intensity of peak 2 is lower than that of peak 1 in the spectra for Flu-HSA complex (Figure 7 and Table 4) with a ratio of 45.0 %. It means that the fluorescence quenching of HSA by Flu on peak 2 is greater than on peak 1. We could make out that the interaction of Flu with HSA induced the

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Journal Pre-proof slight unfolding of the polypeptides of protein, which resulted in a conformational change of the protein, increasing the exposure of some hydrophobic regions which were buried previously. Circular dichroism (CD) spectra was used to further confirm the influence of FLu on the secondary structure of protein. As shown in Figure 8, the binding of Flu to HSA caused increasing in negative ellipticity only at all wavelengths of the far-UV CD without any significant shift of the peaks. The results indicated that Flu induced a

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slight increase in the α-helix structure content of the protein. In addition, the CD

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spectra of HSA in presence and absence of Flu are similar in shape, indicating that

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structures of HSA are predominantly α-helicity in two conditions. From the above

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result, it can be deduced that Flu caused a conformational change on HSA by enhancing helical stability of protein.[26] The calculated results exhibited an increasing

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of α-helix structures (from 56.1 to 57.2%), while the content of β-strands (from 7.4%

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to 6.9%), turn (from 19.3% to 19%), and unordered structures (from 17.2 to 16.9%) reduced at molar ratio Flu/HSA of 10:1, respectively (the content of α-helix, β-sheet

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and other secondary conformation were all shown in Table 5). The increasing percentage of protein α-helix structure indicated that Flu was bound with the amino acid residues of the main polypeptide chain of protein and destroyed their hydrogen bonding networks.[26] That is to say, Flu interacted with HSA by forming new complex, and enhanced HSA stability by enhancing the secondary conformation of HSA.[42-44] Molecular docking technology often be used to predict and analysis the affinity and binding orientation between small molecules to biological macromolecules.[45,46] In this study, the binding mechanism of leukemia drug Flu and HSA was predicted by this method. It was proved that HSA has two main binding sites (site Ⅰ and siteⅡ).[47]

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Journal Pre-proof The results of molecule docking were shown in Figure 9. According to the lowest energy configuration of Flu and HSA, The binding energy of Flu and HSA is -11.15 kcal/mol. It can be seen that Flu and HSA are more likely to act at site I through hydrogen bonds interaction. There were 15 major amino acid residues around ligand in the 5nm range, as shown in Figure 9 (b): GLN194, ALA196, VAL455, TYR452, GLU450, LEU481, TRP214, LEU198, LYS199, HIS242, SER202, PHE211, LYS212, ALA210, LEU347. There were hydrogen bonds forming between the hydroxyl group

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on Flu and the oxygen atoms on the main chain of VAL455 and TYR452 (black

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dotted line), and between the amino group of Flu and the oxygen atoms on the main

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chain of LYS212 (black dotted line). And a pigeon-product pileup was formed by the

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aromatic ring part of Flu and TRP214 indoles ring. Therefore, Flu could change the

4. Conclusion

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microenvironment of HSA and interfere its internal fluorescence.

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The interaction mechanism between Flu and HSA has been investigated in this study using different optical techniques and molecular docking technique. The results

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suggested that Flu bond to HSA through a static quenching procedure. The interaction was both enthalpically and entropically driven. Meanwhile, the hydrophobic forces and hydrogen bonds played a major role in the binding process. Additionally, the binding process was spontaneous. For competition displacement, the binding site of Flu on the protein is at site I in subdomain ⅡA. Experimental results also showed that the binding induced a conformational change of HSA, which was further proved by the results of three-dimensional fluorescence spectra and CD spectra. These changes indicated that the biological activity of HSA was weakened in the present of Flu. According to the lowest energy configuration of Flu and HSA, The binding energy of Flu and HSA is -11.15 kcal/mol.

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Conflict of interest There are no conflicts to declare.

Acknowledgements This work was supported by the National Natural Science Foundation of China [21873075, 21503283, 41503067]; Natural Science Foundation of Hubei Province

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[2015CFC873]; Nature Science Foundation of South-Central University for

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Nationalities [XTZ15016, BZY18002]; Fundamental Research Funds for the Central

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Universities and South-Central University for Nationalities [CZQ13003, CZW15074,

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CZW15109].

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Journal Pre-proof Tables Table 1. Stern-Volmer quenching constants (KSV) of the HSA-Flu complex at different temperatures Table 2. Binding constant (Ka) and thermodynamic parameters of HSA-Flu complex. Table 3. The binding constants of competitive experiments of HSA-Flu system Table 4. The characteristic parameters of three dimensional fluorescence spectra Table 5. Secondary structure of Flu-bound HSA determined by SELCON3

Table 1. Stern-Volmer quenching constants (KSV) of the

Ra

298

1.488

0.999

304

1.121

0.999

0.004

310

0.727

0.002

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0.999

S.D.b

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KSV(×104)

0.006

The correlation coefficient; b The standard deviation for the

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T/K

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HSA-Flu complex at different temperatures

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KSV values

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Table 2. Binding constant (Ka) and thermodynamic parameters of HSA-Flu complex Ka(×104)

R

298

1.637

0.999

304

1.596

0.999

310

1.552

0.999

ΔS

ΔG

-1 -1 -1 （J·mol K ） （kJ·mol ）

(kJ·mol-1)

-24.04 -3.41

69.22

-24.45 -24.87

ro

of

HSA-Flu

ΔH

T/K

sample

KSV(×103)

HSA only

8.417

0.998

HSA-warfarin

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Table 3. The binding constants of competitive experiments of HSA-Flu system

0.992

8.552

0.996

-p

R

lP na

HSA-ibuprofen

1.297

Table 4. The characteristic parameters of three dimensional fluorescence spectra

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Systems and parameters

Peak position

HSA

(λex/λem, nm/nm)

Relative intensity (IF) Peak position

HSA-Flu

(λex/λem, nm/nm) Relative intensity (IF)

Fluorescenc e peak one

Fluorescence peak two

280.0/342

231.0/340.0

143.62

75.53

280.0/342.0

231.0/340.5

122.57

41.98

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Table 5. Secondary structure of Flu-bound HSA determined by SELCON3 Molar ratio [Flu]:[HSA]

α-Helix (%) a

β-Sheet (%)

Turn (%)

Unordered (%)

H(r)

H(d)

S(r)

S(d)

0:1

27.7

28.4

3.1

4.3

19.3

17.2

1:1

27.6

28.9

3.1

4.2

19.2

17

3:1

27.6

29.2

3

4.2

19.2

16.8

10:1

27.8

29.4

2.8

4.1

19

16.9

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“r” and “d” represent “ordered” and “disordered”, respectively.

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Journal Pre-proof Figure 1. (a) Absorption spectra of Flu only in PBS; (b) Absorption spectra of HSA-Flu system. [cHSA = 2×10−6 mol/L, cFlu /(10-6 mol/L, the concentrations of Flu from a to h: 0-7×10−6 mol/L at pH = 7.4, a-h: 0; 1.0; 2.0; 3.0; 4.0; 5.0; 6.0; 7.0.)]

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Figure 2. The absorption spectra of HSA-Flu system. (a) HSA; (b) HSA-Flu; (c) Flu; (d) the − − difference absorption between HSA-Flu and Flu. (cHSA = 2×10 6 mol/L, cFlu = 2×10 6 mol/L at pH= 7.4) Figure 3. (a) RLS spectra of HSA in the presence of various concentrations of Flu. [cHSA = 2.0×10-6 mol/L; cFlu/(10-6 mol/L, the concentrations of Flu from a to i: 0-8×10−6 mol/L at pH = 7.4, a-i: 0; 1.0; 2.0; 3.0; 4.0; 5.0; 6.0; 7.0; 8.0)]; (b) Stern–Volmer plots of HSA titrated with Flu at different temperatures. Figure 4. The modified Stern-Volmer plots of Flu and HSA at different temperatures. The inset is the van’t Hoff plot in PBS, pH = 7.4. Figure 5. (a) Fluorescence transients decay spectra of HSA; (b) Fluorescence transients decay spectra of HSA-Flu system. Figure 6. Site marker competitive displacement experiments of HSA-Flu system in the presence of warfarin and ibuprofen, respectively. Figure 7. (a) 3D fluorescence spectra of HSA; (b) 3D fluorescence spectra of HSA-Flu system. (cHSA = 2×10-6 mol/L; cFlu = 33.3×10-6 mol/L) Figure 8. Circular dichroism spectra of HSA in the absence and presence of Flu. The molar ratios of Flu to HSA are from a to d. [cHSA = 2×10-6 mol/L; cFlu /(10-6 mol/L; the concentrations of Flu from a to d: 0-20×10−6 mol/L at pH = 7.4, a-d: 0; 2.0; 6.0; 20.0)] Figure 9. Molecular docking studies of interaction. (a) schematic diagram of HSA-Flu system; (b) detailed view of the optimum poses of the lowest binding energy for the complexes with HSA. The hydrogen bonds between the complexes and HSA are represented using dark dashed lines.

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Journal Pre-proof Declaration of competing interest statement

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There are no conflicts to declare.

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Author statements Author contributions

Jiawen Lei: Data curation Fan Yu: visualization, Investigation Ke Chen: Methodology

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Yi Liu: Supervision, Writing- Reviewing and Editing

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XiaoLe Han: Conceptualization, Methodology, visualization, Supervision, WritingReviewing and Editing Hao Hao: Investigation, Data curation, formal analysis, Writing-Original draft preparation QingYu Li: Investigation, Validation, formal analysis Chenyin Liu: Investigation, Data curation,formal analysis

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Tao Huang: Supervision, Writing- Reviewing and Editing

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Journal Pre-proof Graphical abstract We studied the interaction between Flu and HSA with different spectroscopic methods and molecule docking. The internal fluorescence of HSA was quenched by Flu through static quenching mechanism. Flu bound with HSA and formed a new complex. The interaction between HSA and Flu induced the conformation change of HSA. Flu enhanced the stability of HSA by increasing the second conformation of

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HSA.

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Journal Pre-proof Highlights 1. The anticancer drug Flu could bind to HSA at site Ⅰ, and formed a new compound at ground state. 2. Flu quenched the internal fluorescence of HSA. This behavior is consistent with static quenching mechanism. 3. The hydrophobic forces and hydrogen bonds played major roles in the binding

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process between Flu and HSA. Additionally, the binding process is spontaneous.

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4. The interaction of Flu with HSA induced the conformational change of the protein,

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Flu enhanced HSA stability by enhancing the second conformation of HSA .

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9