摘要：本文通過(guò)熒光光譜、酶解和分子對(duì)接等技術(shù)研究了單寧酸（TA）與人血清白蛋白（HSA）之間的仿生作用力和分子動(dòng)力學(xué)模擬。結(jié)果表明，TA與HSA的相互作用是一個(gè)典型的兩階段反應(yīng)：快反應(yīng)階段（RRS）和慢反應(yīng)階段（SRS）。在25 ℃時(shí)，結(jié)合常數(shù)分別為3.16×105 L·mol-1和3.55×108 L·mol-1。熒光光譜表明TA對(duì)HSA的猝滅機(jī)理是靜態(tài)猝滅效應(yīng)，且疏水力在TA與HSA相互作用過(guò)程中起著重要作用?；贔?rster 非輻射能量轉(zhuǎn)移理論，確定TA 的結(jié)合位點(diǎn)位于亞結(jié)構(gòu)域A 的疏水區(qū)域，TA 與人血清白蛋白Trp212 之間的距離分別為2.21 nm（RRS）和1.97 nm （SRS）。疏水探針和酶解實(shí)驗(yàn)證實(shí)TA與HSA之間的相互作用分為兩個(gè)獨(dú)立的過(guò)程，包括快速反應(yīng)中的瞬時(shí)識(shí)別并耦合及慢速反應(yīng)中的再匹配。本研究驗(yàn)證了TA與HSA的相互作用力，并闡明其反應(yīng)過(guò)程，為植物多酚與蛋白質(zhì)互作模式的研究提供了參考依據(jù)。

關(guān)鍵詞：快反應(yīng)；慢反應(yīng)；熒光光譜；分子對(duì)接

中圖分類(lèi)號(hào)：Q946 文獻(xiàn)標(biāo)志碼：A 文章編號(hào)：0253-2395（2024）05-1086-11

0 Introduction

Tannic acid （TA） is a hydrolysable tannin polyphenolcompound derived from plants. It forms interactionswith proteins and polysaccharides throughhydrogen bonding， electrostatic， coordinative bonding，and hydrophobic interactions. TA is widely utilizedfor its anti-cancer， anti-bacterial， and food antioxidantproperties[1-3]. The ability to interact andbind with proteins is considered as the most crucialfactor in generating biological effects[4-5]. TA isfound in various foods， red wine and tea. However，tannins can also reduce the permeability of the humanintestinal wall and affect the digestion[6]. Additionally，the complex formed by tannins and proteinsinterferes with the absorption and utilization ofprotein nutrients[7]. TA represents the fundamentalfunctional unit of plant tannins， with its primarystructure being glucose oligohydroxyl. Therefore， usingtannic acid as a model substance to study its interactionmechanism with proteins is representativeand universally applicable. Human serum albumin（HSA） is a monomeric protein comprising 585amino acids， organized into three α -helical domains（I， II and III） containing two subdomains （A and B）within each α -helical domain[8]. One of the most importantbiological functions of albumins is their abilityto transport endogenous and exogenous substances[9-10]. HSA serves as a model protein which isextensively used to study interactions with smallmolecules[11-12]. It is also employed as a standardproduct to eliminate the influence of non-uniformstructure.

Various methods have been employed to analyzethe tannin-protein complexes， includingHPLC[13]， UV-Vis[14]， turbidimetry， electrophoresis[15]，and infrared imaging spectroscopy[16-17]. Several modelsand theories have been reported and discussed，such as the hydrophobic action and multi-point hydrogenbonding theory[18]， the selective rule oftannin-protein mutual recognition， the glove-hand actionmode[19]， the tannin-protein co-precipitation andprecipitation mechanism[20]. In recent years， it hasbeen proposed that the plant polyphenols approachthe protein molecule surface through hydrophobicinteractions， followed by the occurrence of multipointhydrogen bonding[21]. The extent of binding isinfluenced by the structure of polyphenols[22-23]. Therapid binding between polyphenols and peptidesleads to the conformational changes in the synthesizedpeptides[24]. Among these theories， \"the hydrophobicaction and multi-point hydrogen bondingtheory\" is the most popular， although research on itsreaction process is limited. Therefore， this workaims to supplement a possible mechanism for the reactionprocess.

It is important to explore the interaction mechanismbetween TA and biological proteins to enhancenutrient absorption and promote human health. Planttannins have glucosamine skeletons with hydroxylgroups attached to the periphery of different sugars.The structure of TA is the simplest， and if it undergoestwo reaction processes， it is likely that otherpolyhydroxy tannins also undergo them. In fact， it isthe interaction between hydroxyl groups and proteins.In this study， the binding process of TA to HSAwas analyzed using fluorescence spectroscopy， dockingand molecular dynamics simulation. The relationshipbetween the protein hydrophobic domain andpolyphenols was investigated using the hydrophobicprobe 2-P-toluidinylnaphthalene-6-sulfonate （TNS）and enzymatic hydrolysis， revealing that this interactionwas based on the tertiary structure of protein. Apossible mechanism of \"conformational rematch\" forthis interaction is proposed in this work.

1 Materials and Methods

1. 1 Apparatus

All fluorescence analysis was conducted usingthe Hitachi F-4500 spectrofluorometer. The absorp‐tion spectrum was obtained by using the HitachiUV-2010 spectrophotometer. The pH values weremeasured by using the Inolab digital pH meter.

1. 2 Materials and main reagents

Hepes （N-（2-hydroxyerhyl） piperazine-2-erhanesulfonicacid） was obtained from Kermel ChemicalReagent Co， Ltd. （Tianjin， China）， while TA andTNS were acquired from Sigma Co， Ltd. （St. Louis，MO， USA）. Additionally， HSA was sourced fromSolarbio Co.， Ltd. （Beijing， China）. All of chemicalsused in this study were of analytical grade.

1. 3 Docking procedure

The crystal structure of HSA （1N5U） was obtainedfrom the Protein Data Bank （https：//www.rcsb.org/pdb）. The original crystal structure of HSA wasprocessed using Auto Dock Tools 4.2.6. This involvedremoving non-polar molecules， adding all hydrogenatoms， and preserving the original charge ofOVA before exporting it as a. pdbqt file. The 3Dstructure of TA was drawn using ChemOffice 18.0，which was further processed with Auto Dock Tools1.5.6 to Generate a. pdb file for the docking study.The protein-ligand complexes with the lowest energieswere selected as experimental models. Thedocking conformations of these complexes werethen examined using PyMol.

1. 4 Molecular dynamics simulation

A molecular dynamics simulation was conductedaccording to the procedure reported by Faniet al[25] with some modifications. A 10 ns moleculardynamics simulation was performed using the Gromacs2019 software package. The CHARMM36force field was used， and the topology file andsmall molecule charge were generated using thewebsite https：//cgenff. paramchem. org/. A dodecahedronbox was selected and filled with solvent beforeundergoing energy minimization and optimization.Following the energy minimization process， anisothermal-isobaric ensemble （NPT） simulation wasperformed. This involved applying a constant pressureof 101.325 kPa at a constant temperature of300 K for a duration of 100 ps. Finally， a 10 ns moleculardynamics simulation was performed.

1. 5 Preparation of samples

A 2 mL solution of human serum albumin（HAS） with a concentration of 5×10?6 mol·L?1 wassubjected to titration by a solution of tartaric acid（TA） at a pH of 7.4 at room temperature. A 10 mLaliquot of HSA solution were mixed with an appropriateamount （0～250 μL） of 1×10?6 mol·L?1 TA solutionin volumetric flask to the final concentrationof 5×10?6 mol·L?1. Then it was shaken and equilibratedfor 24 h at 25 ℃ and 37 ℃， respectively.

For the fluorescence measurements， an excitationwavelength of 295 nm was used with the excitation，and emission slits set at 10 nm each. Thefluorescent spectrum was recorded in the range of300 to 600 nm. All solutions were prepared using0.01 mol·L?1 Hepes buffer solutions （pH 7.4） tomaintain the ionic strength， a 0.1 mol·L?1 sodiumchloride （NaCl） solution was employed. Furthermore，all solutions were prepared using Ultra-purewater obtained from a Milli-Q water purificationsystem and stored at 4 ℃.

1. 6 SDS-PAGE analysis

HSA and TA were mixed with ratio of 1∶ 0.2for 1 h， 3 h， 9 h and 16 h， and the compounds wereincubated with 0.1 mg·mL?1 trypsin at 37 ℃ for 40min. Then the incubation mixtures were separatedby Sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） with a separating gel composingof 10% acrylamide[26]. Then the gels werestained in the fluorescent gel stain solution， andwere washed with sterile deionized water， photographedand analyzed.

1. 7 Statistical analysis

Analyses of variance and regression equationswere performed using SASv9.。2 Results and Discussion。2. 1 Molecular docking and co-acting forces inthe HAS-TA complex

It has been reported that， the II A and III A inHSA are the most common binding sites[27-28]. The sta‐bility of a ligand-protein complex is directly correlatedwith its binding affinity， with lower energy valuesindicating stronger stability. As depicted in theFig. 1（b）， the conformation with the lowest bindingenergy （-12.6 kcal·mol?1）， which suggested the formationof a stable complex between TA and HSA.

Given the presence of numerous hydroxylgroups in TA， hydrogen bonding plays pivotal rolein the interaction between TA and HSA. Seventeenhydrogen bonds were identified in the dockingmodel （Fig. 1（a））， including Tyr148 （2.6 ?）， Tyr150（2.4 ?）， Arg160 （2.8 ?）， Glu188 （2.8 ?）， Lys199（1.8 ?）， Trp214 （2.5 ?）， Arg218 （2.3 ?）， Gln221（2.3 ?）， Arg222 （2.2 ?）， Arg257 （2.6 ?）， His242（2.0 ?）， Ala261 （2.5 ?）， Glu292 （2.7 ?）， Asn295（2.4 ?）， Lys444 （2.6 ?）， Asp451 （1.9 ?）， andSer454 （2.3 ?）. Additionally， π - π stacking occurredin TA harboring rich benzene rings and HSA containingphenyl amino acids （Tyr452， Phe156，Phe157， Phe149， Tyr150， Phe223， Phe211）. In summary，the binding of HSA-TA is facilitated by hydrogenbonding， π - π accumulation and hydrophobicinteractions， ultimately leading to structural modificationsin HSA.

2. 2 Molecular dynamics simulation

The dynamics analysis of the binding betweenTA and HAS was conducted using molecular dynamicssimulation. The conformational changes ofHSA were assessed by calculating parameters suchas root mean square deviation （RMSD）， root meansquare fluctuation （RMSF） and radius of gyration（Rg）. The RMSD is a statistical measure that describesconformational deviations， and reflects thestability of the system. On the other hand， RMSF isemployed to investigate the flexibility of protein residuesby means of residue analysis. As shown in Fig.2（a）， the RMSD value exhibited the fluctuationsthroughout the whole simulation process of HSA-TAcomplex， suggesting the formation a stable complexand a tendency towards system stability. However，the RMSF value of amino acid residues 120-130，180-200 and 290-310 in the HSA-TA system wassignificantly higher than that in the HSA system， indicatingthat the binding of HSA-TA had a greaterimpact on amino acid residues at these sites （Fig. 2（b））. Additionally， the Rg value was utilized to measurethe compactness of the HSA structure. A higherRg value indicates， a looser structure. As illustratedin Fig. 2（c）， the Rg value of the HSA-TA complex，generally exceeded that of the HSA， implying thatthe interaction between TA and HSA led to the exclusionof amino acid residues， thereby disrupting thespiral and folded structure， and resulting in an expandedsystem.

2. 3 Influence of reaction time

Previous studies have reported that the interactionsbetween polyphenols such as ellagic acid （EA），epigallocatechin gallate （EGCG）， epicatechingallate（ECG） and gallic acid （GA） and HSA involve onlyone rapid reaction stage （RRS）[9，29]. In this study， weaimed to investigate the stability of the reactiontime between TA and HSA， by monitoring the fluorescencespectrum curve over a period of 24 h. Asshown in Fig. 3（a）， the fluorescence of the HSA-TA complex exhibited changes over time， revealing thepresence of two distinct reaction stages： the RRSand the slow reaction stage （SRS）. Upon addition ofTA to the HSA solution， an immediate decrease influorescence intensity occurred， followed by agradual decrease after a stabilization period of 1.5hours， eventually reaching equilibrium.

2. 4 Fluorescence spectrum analysis of HSA-TA

Titration and interaction experiments were conductedto analyze the interaction process betweenTA and has. It was observed that TA exhibitedminimal fluorescence at 345 nm when excited at awavelength of 295 nm. Therefore， the emission fluorescencedetected at 345 nm solely originated fromHSA intrinsic contribution unaffected by TA.

As shown in Fig. 3（b）， the fluorescence ofHSA at 345 nm was quenched by TA gradually， andthe similar result was observed at 37 ℃， which indicatedthat the conformation of HSA might havebeen changed. Additionally， a red shift in the maximumemission of wavelength of HSA was foundupon the addition of TA （Fig. 3（c））. Notably， the impactof TA on HSA fluorescence was observedwithin a range of specific concentration. Specifically，it was observed that the protein endogenousfluorescence was quenched when the TA concentrationwas below 4×10?6 mol·L?1. Conversely， a significantred shift in the protein's fluorescence spectrumpeak occurred when the concentration exceeded4×10?6 mol·L?1. These findings indicatethat the binding process of TA resulted in a transitionof the tryptophan environment from hydrophobicto hydrophilic， suggesting a significant alterationin protein conformation dependent on TA concentration.

2. 5 Quenching mechanism

In general， the differentiation between dynamicand static quenching is based on the regulation oftemperature. Therefore， the fluorescence spectra ofTA to HSA quenching were examined at the temperatureof 25 ℃ and 37 ℃. Subsequently， the Stern-Volmer curves of TA with HSA were constructed. Itwas observed that the Stern-Volmer plots were exhibitedlinearity， with the slopes of decreasing asthe temperature increased. Furthermore， the resultsobtained after a 24 h of interaction were consistentwith those obtained through titration methods. Thispreliminary evidence proved the existence of astatic quenching interaction between TA and HSA.

To further elucidate the mechanism of fluorescencequenching， the fluorescence quenching data attemperatures of 298 K and 310 K were analyzed usingthe classical Stern-Volmer equation[30]， as depictedin Figure 4（a）.

F0／F = 1 + Kq τ0 [ Q ]= 1 + KSV [ Q ] ， （1）

where， F0 and F are the fluorescence intensity in theabsence and presence of quencher （TA）， respectively.Kq， Ksv， τ0 and [Q] are the quenching rate constant ofthe biomolecule， the dynamic quenching constant，the average lifetime of the biomolecule withoutquencher and the concentration of quencher， respectively.Because the fluorescence lifetime of the biomacromoleculeis 10?8 s[31]， Ksv is the slope of the lin‐ear regression equation （Fig. 4（a））. According toequation （1）， the quenching constant Kq can be calculatedand is listed in Table 1.

2. 6 Binding constant and binding site analysis

For static quenching， the following equationwas employed to calculate the binding constant andthe number of binding sites[33]：

lgF0 - F／F = lg KA + n lg [ Q ] ， （2）

where， KA and n are the binding constant and thenumber of binding sites， respectively. Thus， a plotof lg [（ F0-F ）/F ] versus lg[Q] can be used todetermine KA and n in Fig. 4（b）.

The data of KA and n values were presented inTable 2， indicating the presence or approximatelyone binding site during the RRS， and nearly two binding sites during the slow reaction stage. However，the temperature demonstrated minimal influenceon the binding constant （KA） of TA and HSA.Notably， the binding constants and binding sites observedduring the 24-h interaction （SRS） werehigher than those observed during titration （RRS）（Table 2）， signifying an increased binding strengthbetween TA and HSA following the slow reactionstage. This suggests that HSA is more readily ableto bind additional TA molecules after the initialbinding event.

2. 7 Thermodynamic evaluation of the interac?tion between TA and HSA

The interactions between a drug and a biomoleculeare primarily comprised of weak forces， includinghydrogen bond formation， van der Waalsforces， electrostatic forces， and the hydrophobic interaction.It has been noted that the determination ofthese interaction forces can be achieved throughconsideration of the thermodynamic parameters. Specifically，△H gt; 0 and △S gt; 0 implies hydrophobicinteraction; △H lt; 0 and △S lt; 0 reflects the van der Waals force or hydrogen bond formation[34].The thermodynamic parameters can be obtainedfrom the following equations and the results wereshown in Table 3：

lnk2／k1= （ 1／Τ1- 1／T2 ）ΔH／R ， （3）

ΔG =-RT ln K ， （4）

ΔG = ΔH - TΔS . （5）

It can be concluded that the interaction betweenTA and HSA is thermodynamically spontaneous.The thermodynamic parameters （ ΔH and ΔS）as shown in Table 3， are higher than zero， indicatingthat the predominant forces between TA andHSA are mainly hydrophobic forces. However， it'sunavoidable that multi-forces may take part in theinteraction at the same time， owing to the intricatestructure of HSA and TA， which further complementand validate the results from molecular dockingand molecular dynamics simulation analysis.

2. 8 Transferring energy between TA and HSA

According to F?rster non-radiative energy transfertheory[35]， HSA functions as a donor and exhibitsstrong intrinsic fluorescence. The Fig. 4（c） demonstratesthe efficient energy transfer that takes placebetween TA and HSA， due to the considerable spectraloverlap between the fluorescence emission spectrumof HSA （2） and the UV absorption spectrumof TA （1）.

The energy transfer effect is related not only tothe distance between the acceptor and the donor， butalso to the critical energy transfer distance. The relationamong these factors is：

E =R60／R60+ r6 ， （6）

where， r is the distance between the acceptor andthe donor， and R0 is the critical distance when thetransfer efficiency is 50%， in turn， which can be calculatedby：

R60= 8.8 × 10-25 K2ΦNJ ， （7）

where， K2 is the spatial orientation factor of the dipoles，Φ is the fluorescence quantum yield of thedonor， N is the refractive index of the medium， andJ is the overlap integral of the fluorescence emissionspectrum of the donor and the absorption spectrumof the acceptor. So J can be calculated usingthe following formula：

J = ΣF（ λ ） ε（ λ ） λ4Δλ／ΣF（ λ ）Δλ， （8）

where F（ λ ） is the fluorescence intensity of the fluorescentdonor at wavelength λ， and ε（ λ ） is the molarabsorptivity at the acceptor wavelength λ. The energytransfer efficiency is given by：

E = 1 -F／F0， （9）

where， J can be evaluated by integrating thespectra in Fig. 4（c） and Eq. （8） for λ= 260-560 nmand can be 1. 11×10-14 cm3·L·mol-1. Under these ex?perimental conditions， we found a characteristic dis?tance of R0 = 2. 60 nm， using K2 = 2/3， N = 1. 336，Φ = 0. 15［36］. The energy transfer effect E is 0. 73（RRS） and 0. 84 （SRS） from Eq. （9） and the maxi?mum distance between TA and tryptophan residue inHSA， Trp212 was 2. 21 nm （RRS） and 1. 97 nm（SRS）. This confirms that the energy transfer be?tween TA and HSA contributes to the decrease ofHSA fluorescence intensity. The binding site for TAmay be in a hydrophobic region in the sub-domainIIA of HSA. After interaction for 24 h， the distancebetween TA and tryptophan in HSA decreased， indi?cating that the combination of TA and HSA wasstrengthened. It clearly suggested that the slow reac?tion stage is more stable than the reaction stage.

2. 9 Analysis of the binding process using hydro?phobic fluorescence probes

TNS is considered as one of most importanthydrophobic fluorescent probes in the field of proteinresearch[37]. Upon excitation at a wavelength of322 nm， the HSA-TNS complex exhibited a strongintensity in fluorescence with the maximum emissionat about 440 nm （Fig. 5）. This observation indi‐cates that TNS binds to the hydrophobic region ofthe protein. Furthermore， the addition of TAquenched the fluorescence of HSA-TNS， which affectedthe hydrophobic environment of TNS.

In addition， the fluorescence was rapidlyquenched when the ratio of TA： HSA-TNS wasranged from 0-1， followed by a gradual decline beyondthat ratio. This phenomenon indicated that theinteraction between TA and HSA does not completedin one step. TA and HSA do not reach thegoal in one step， and it has a slow process of rematching，which was consistent with the previousreport[38].

2. 10 Interaction analysis of TA and HSA using the enzymolysis method

To further investigate the interaction betweenHSA and TA， we employed the SDS-PAGE analysismethod. The HSA-TA complex formation processwas enzymatically treated with trypsin to assess thedigestibility of trypsin with respect to the durationof interaction between TA and HSA. As depicted inFig. 6， after 16 h of interaction between HSA andTA， trypsin basically could not digest the compoundsformed by HSA and TA， which suggestedthat the interaction between TA and HSA was notachieved instantaneously， and the protein complexstructure became more stable after the RRS andslow reaction. In a study conducted by Adrian et al.In 2022 tannins were found to rapidly bind to shortpeptides and synthetic peptides resulting in conformationalchanges in the synthetic polypeptides. So，they concluded that the mechanism of interactioncould be TA combined instantaneously with protein[39]. However， after the binding of TA to protein，the conformation of the protein changed graduallyto make the binding sites tighter， which could generatea more stable complex. It revealed that rematchingof hydrogen bond donors and acceptors， as well as hydrophobic regions was occurring between TAand the protein， and it was a relatively slow process.

3 Conclusion

In this study， we investigated the nanoscale bioinspiredinteraction analysis of TA and HSA usingfluorescence spectroscopy， molecular docking anddynamic modeling. Our findings highlight the significantrole of hydrogen bonding during the interactionprocess between TA and HSA. We observedthat the binding ability is similar to that of casein，which containing about 16% proline， although HSAcontains about 4% proline. Furthermore， we observedan enhancement in binding ability with an increasein the molecular weight of polyphenol. In addition，our results suggest that the selectivity of TAtowards proteins is influenced by variables such asthe molecular weight of proteins， proline content，protein structure and pI value. It is worth notingthat while the emphasis has traditionally been onthe composition of amino acids in the primary structureof proteins， our study underscores the importanceof hydrophobic structures formed in the secondaryand higher structure of the protein. The resultsindicate that hydrophobic interactions play apivotal role in this type of interaction， accompaniedby the synergistic effects in the formation of hydrogenbonds. Based on the kinetic model of plantpolyphenols-protein reaction， we propose a two-stepprocess： first， a rapid combination through hydrophobicaction and hydrogen bonding， second， followedby further conformational changes and rematching，resulting in the fluorescence quenching ofthe protein. Notably， this study presents， for the firsttime the \"conformational mismatch\" hypothesis ofthe interaction between TA and HSA， providingvaluable insights into the interaction between polyphenoliccompounds and protein biomolecules.

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基金項(xiàng)目：山西省教育科學(xué)“十四五”規(guī)劃課題（TY-230016）；山西省哲學(xué)社會(huì)科學(xué)項(xiàng)目（2023YJ148）