A novel method for the study of molecular interaction by usingmicroscale thermophoresis

Yexuan Mao a, Lanlan Yu a,n, Ran Yang a, Ling-bo Qu a,b,nn, Perter de B. Harrington c

a College of Chemistry and Molecular Engineering, Zhengzhou University, Kexue Road, Zhengzhou 450001, PR Chinab School of Chemistry & Chemical Engineering, Henan University of Technology, Zhengzhou 450001, PR Chinac Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Clippinger Laboratories, OHIO University, Athens,OH 45701-2979, USA

a r t i c l e i n f o

Article history:Received 7 May 2014Received in revised form21 September 2014Accepted 28 September 2014Available online 31 October 2014

Keywords:Microscale thermophoresisMolecular interactionBovine serum albuminFluorescein isothiocyanateCompetitive binding study

a b s t r a c t

The fundamental studies for the binding events of protein and its partner are crucial in drugdevelopment. In this study, a novel technology named microscale thermophoresis (MST) was appliedin the investigation of molecular interaction between an organic dye fluorescein isothiocyanate (FITC)and bovine serum albumin (BSA), and the results were compared with those obtained from conventionalfluorescence spectroscopy. The MST data demonstrated that with a short interaction time, FITC showed ahigh binding affinity for BSA by weak interaction instead of labeling the protein. By using competitivestrategies in which warfarin and ibuprofen acted as the site markers of BSA, FITC was proven to mainlybind to the hydrophobic pocket of site II of BSA compared to site I of BSA. Except for the binding affinity,MST also provided additional information with respect to the aggregation of BSA and the binding of FITCto BSA aggregates, which is unobtainable by fluorescence spectroscopy. This work proves that MST as anew approach is powerful and reliable for investigation of protein–small molecule interaction.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

In vivo, cellular signaling involves a series of recognition events,leading from an external stimulus to a cellular response. Each recog-nition event is the binding between a protein and a partner thatranges from a small-molecule ligand to a macromolecular com-pound, which is the center of biological and biochemical processes inliving organisms [1–3]. Notably, the interactions between biomole-cules such as protein-protein and protein–small molecule with awide range of affinities, have generated great interests for funda-mental investigations in vivo and the development of targets fordrugs [4–6]. Moreover investigation on these recognition eventshelps elucidate the structural nature of bioaffinity of drugs and theirbinding sites. Equilibrium binding experiments are extensively app-lied in the investigation of binding and competitive binding eventsin biological systems [7]. Recently, competitive binding behaviorshave aroused more concerns to investigate the interactions betweenproteins and their ligands.

Up to present, a variety of powerful tools have been developedto study biomolecular interactions, such as absorption spectroscopy,

circulardichroism [8], fluorescence spectroscopy [9], isothermal titra-tion calorimetry (ITC) [10,11], fluorescence polarization (FP) [12,13],surface plasmon resonance (SPR) [14,15], dynamic light scattering(DLS) [16] and so on. These approaches are useful and convenient forinvestigating biomolecular interaction, but also have some disadvan-tages. For instance, fluorescence quenching or fluorescence correla-tion spectroscopy can be applied to analyze equilibrium constantsof the interactions. However, these methods are only suited for afew interactions, as they crucially rely on molecular size and size-change upon binding parameters, the relative position of fluoro-phores to each other [17], and consumption larger quantities ofsample or require extensive data analysis [18].

Although ITC is the most common nonfluorescent method thatcan monitor and record a biological binding process by micro-calorimetry, obtaining the complete thermodynamic data requireslong acquisition times and a relatively large amount of sample toobtain an adequately heat signal [19,20]. FP is a fully automatedhigh-throughput analysis, but the limited lifetimes of the fluor-escent dyes and the molecular weight change on binding hinderits applicability and sensitivity.

Although SPR is based on the changes of the physical opticsof bound molecules and has relatively high sensitivity, it is notapplicable in free solution because it relies on immobilizing oneof the binding partners onto a thin noble metal film. Nonspecificbinding of sample components onto the metal film may alsointerfere with the measurement. DLS is a label-free analytical

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Talanta

http://dx.doi.org/10.1016/j.talanta.2014.09.0380039-9140/& 2014 Elsevier B.V. All rights reserved.

n Corresponding author. Tel.: þ86 37167756886; fax: þ86 37167756886.nn Corresponding author at: The College of Chemistry and Molecular Engineering,

Zhengzhou University, Kexue Road, Zhengzhou 450001, PR China.Tel: þ86 13525561266.

E-mail addresses: yulanlan@zzu.edu.cn (L. Yu), qulingbo@zzu.edu.cn (L.-b. Qu).

Talanta 132 (2015) 894–901

technology, using the relationship of time-dependent fluctuationsof light scattered by molecules in solution, applied in free solutionbinding investigations. However, this approach requires an obvi-ous difference in the dynamic radius (rH) of the unbound partnersrelative to the complex. Considering rH scales with the cube root ofthe molecular volume, the sensitivity of large particles is higherthan that of smaller particles [21,22].

Recently, a new method microscale thermophoresis (MST)technology has emerged, monitoring the directed movement offluorescently labeled molecules through temperature gradients.Distinguished from the other binding measurements, MST is ahighly sensitive probe for many kinds of binding-induced interac-tions such as molecular size, charge, hydration shell or conforma-tion [37]. Typically, the size, charge, and hydration entropy obtai-ned by the thermophoresis of the fluorescently labeled mole-cules do not differ from those obtained from the unlabeled com-plexes. Furthemore, MST possesses several advantages includingeasy implementation, faster speeds (about 1 min), and lowersample requirements (e.g., microliter volumes). In addition, a widevariety of buffer solutions can be used and there is no practicallimitation on molecular size or weight for the binding partners.

In this study, the interaction between fluorescein isothiocya-nate (FITC) and albumin bovine serum (BSA) was investigated byusing MST, which provides a new method for biomolecularinteraction and proves the feasibility of MST for biomolecularinteraction studies. FITC is currently the most widely used fluor-escein dye for protein labeling, imaging and so forth. In thebiological pharmacology field, serum albumin (SA) protein is themost abundant soluble protein found in blood plasma [23], whichcan combine with many kinds of endogenous or exogenouscompounds such as amino acids, fatty acids, drugs, and manyother small molecules [24,25]. BSA is an SA protein often used inbiological investigations because it has a 76% sequence homologywith human serum albumin.

The binding regions of BSA have been previously indentified bycrystallography [26,27], which contain three structurally similardomains (I, II, and III) with each domain including two sub-domains.Sudlow's sites I and II are known as the common high affinity regions.The two tryptophan residues Trp-134 and Try-212 of BSA are locatedin sites I and II, respectively [28,29]. An anticoagulant drug warfarinand an anti-inflammatory drug ibuprofen have been demonstrated asstereotypical marker ligands for sites I and II, respectively [30].Therefore by using binding studies, it is possible to identify thelocation of BSA that binds FITC. In this study, MST is demonstratedas a new approach for biomolecular interaction and the results arecompared with those from conventional fluorescence spectroscopy.

2. Experimental

2.1. Apparatus

Fluorescence spectra were recorded on 970CRT spectrofluorom-eter (Shanghai, China). The width of the excitation and emission slitswere 5.0 nm and 10.0 nm, respectively. The sensitivity was 3.

The binding affinity study was investigated on the microscalethermophoresis instrument (NanoTemper Tchnologies GmbH, Munich,Germany). The value of Kd was calculated by NT ANALYSIS SOFTWAREprovided by NanoTemper Technologies GmbH. All the pH values weremeasured with a PHS-3C precision pH meter (Leici Devices Factory ofShanghai, China), which was calibrated with standard buffer solutionseach day.

2.2. Materials and reagents

BSAwas purchased from Roche Pharmaceuticals, Ltd. (Shanghai).Warfarin and ibuprofen were purchased from the National Institute

for the Control of Pharmaceutical and Logical Products (China). FITCwas purchased from Aladdin Reagent (Shanghai). Phosphate buffersolution (PBS) was prepared at a pH of 7.4 and a concentrationof 10 mmol L�1 and was used in all experiments. All chemicalsand reagents were of analytical grade and used without furtherpurification.

2.3. Labeling procedure

BSA was dissolved in PBS (pH 7.4, 0.01 mol L�1) with aconcentration of 20 mg mL�1, and FITC was dissolved in dimethylsulfoxide (DMSO) with a concentration of 1 mg mL�1. BSA wastitrated by FITC slowly with a final mass ratio of 1:0.15 (BSA: FITC).The mixture of BSA and FITC was incubated at 277 K for 8 h indarkness. Subsequently, 30 μL of NH4Cl (5.0 mol L�1) was added tothe mixture with a final concentration of 50 mmol L�1 to quenchthe reaction. Unreacted FITC and the FITC-labeled BSA wereseparated on a G-50 Sephadex column with 0.01 mol L�1 PBSeluent. Finally, the concentration of FITC-labeled BSA was deter-mined as 0.9 μmol L�1 with a UV–vis spectrophotometer.

2.4. Theoretical background of MST

MST is a method based on micro-thermal effect of mobility. Thedirectional movements of molecules on a microscopic scale arehighly sensitive to many kinds of binding-induced interaction suchas molecular size, charge, hydration shell or conformation (Thedetails were reported by Seidel et al. [31]). When the liquid isheated locally, the molecules begin to move along the temperaturegradient from high temperature to low temperature. As themovement reaches an equilibrium state, the spatial concentrationdistribution of the liquid can be expressed by the equation:

chotccold

¼ exp �ST T�T0ð Þ½ � ¼ exp �STΔT� �� 1�STΔT ð1Þ

for which chot is molecular concentration in the hot area; ccold ismolecular concentration in the cold area; ST is the Soret coeffi-cient; T0 is the initial temperature; T is the elevated temperature.Although the theory of thermophoresis is nascent, most theoriesconsider that various parameters may influence ST, such as size,charge, hydration layer and solvation entropy. The Soret coefficientmay be expressed by the following equation:

ST ¼AkT

�ΔshydðTÞþβσ2

ef f

4εε0T� λDH

!ð2Þ

for which A represents the molecule's surface area, σeff is theeffective charge, Δshyd is the hydration entropy of the molecule-solution interface, λDH is the Debye-Hüeckel screening length, ε isthe dielectric constant, and β is temperature derivative of ε.

Fig. 1 is a schematic of the MST instrument for signal acquisi-tion for a single capillary that contains the fluorescent species andits binding partner. An infrared (IR) laser is coupled into the lightpath of fluorescence excitation and emission, is focused on thesample to the exact spot where fluorescence emission is detected.The fluorescence within the capillary is excited and measuredthrough the same optical elements. Initially, the infrared laser is offand there is no temperature gradient in the solution. Therefore thefluorescent species is homogenously distributed and a steady fluor-escence signal is acquired. Once the infrared (IR) laser is switched on,the focused spot is heated immediately. The increase of temperatureleads to a strong decrease in fluorescence due to the sensitivity offluorescence quantum yield with respect to temperature, whichcreates an observable temperature jump. Due to thermophoreticmolecular motion, the fluorescence decreases slowly until reaching asteady state when the thermophoretic motion is counterbalanced bythe mass diffusion. Because of the short timescale of the temperature

Y. Mao et al. / Talanta 132 (2015) 894–901 895

jump process (about 100 ms), it can be consequently distinguis-hed from the following slower thermophoretic process. When theIR-laser is switched off, the fluorescence increases suddenly becauseof the decrease in local temperature, which results in a reversedtemperature jump. After a short time of fluorescence recovery, thefluorescence increases gradually until achieving a steady state due toback-diffusion.

The dissociation constant Kd quantifies the binding affinity byanalyzing the change in Fnorm, which is the relative fluorescence(normalized fluorescence). Fnorm is used for quantifying the con-centration of fluorescent molecules and is defined by the followingequation, including both the temperature jump process and thethermophoresis process.

Fnorm ¼ FhotFcold

¼ 1þ δFδT

�ST

� �ΔT ¼ chot

ccoldþδFδTΔT ð3Þ

In the above equation, Fhot is the fluorescence in the hot area andthe Fcold is the fluorescence in the cold area, both of which are theresults from all fluorescent species in solution. When a fluorescentspecies binds to a non-fluorescent ligand and forms a fluorescentcomplex, the thermonphoretic signals of the bound species andunbound species are linearly superposed and described by thefollowing equation

Fnorm ¼ ð1�xÞFnorm;unboundþxFnorm;bound ð4Þ

for which the Fnorm is the total normalized fluorescence, Fnorm,

unbound is the normalized fluorescence of the unbound fluorescentspecies, Fnorm, bound is the normalized fluorescence of the boundfluorescent complex and x is the fraction of the bound fluorescentspecies. By titrating a fluorescent species with a fixed concentrationof a non-fluorescent ligand with varying concentrations and record-ing, Fnorm is recorded versus different concentrations of the non-fluorescent ligand and the data are fitted according to differentmathematical models.

Applying the law of mass action, the binding behavior ofmolecules can be described by a variety of models. The simplestbinding model is the partner bound with its ligand at ratio of 1:1,which can be expressed as

AL3AþL ð5Þ

for which A is the binding partner; L is the titrating ligand; AL isthe bound complex of A and L. Subsequently, the dissociationconstant Kd can be defined as

Kd ¼½A�½L�½AL� ð6Þ

for which [A] is the equilibrium concentration of the free partner;[L] is the concentration of the free titrating ligand; [AL] is theconcentration of bound complex of A and L. As the free equilibriumconcentrations of A and L are not easily known, the analyticalconcentrations of A and L are used instead, which are expressed as:

cA ¼ ½A�þ½AL� ð7Þ

cL ¼ ½L�þ½AL� ð8Þfor which cA is the analytical concentration of A and cL is theanalytical concentration of L. Combining the above Eqs. (6)–(8),the dissociation constant Kd may be expressed as

Kd ¼ðcA�½AL�ÞðcL�½AL�Þ

½AL� ð9Þ

Typically, the binding behavior of A and L is investigated bytitration in microscale thermophoretic experiments. A is a fluor-escent substance with a fixed concentration and L is the titratingligand which is increased in concentration. Hence, the fractionbound of A can be described as:

x¼ ½AL�cA

¼cAþcLþKd�

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðcAþcLþKdÞ2�4cAcL

q2cA

ð10Þ

Combining the Eqs. (4) and (10), the value of Kd can becalculated by NT ANALYSIS SOFTWARE provided by NanoTemperTchnologies GmbH.

2.5. MST experiments

The concentration of FITC stock solution was 0.4 μmol L�1

prepared in PBS (0.01 mol L�1, pH of 7.4) and FITC also acted as ameans of monitoring thermophoretic movement due to its fluores-cence. A serial dilution of BSA (0.02 μmol L�1 to 2.0 mmol L�1) inthe same buffer (PBS pH 7.4) was prepared and mixed with the

Fig. 1. A displays the experimental instrument of MST. The measurements are investigated in capillary. An infrared laser coupled into the light path of fluorescence excitationand emission is focused on the sample to exact spot where fluorescence intensity is measured. B is a typical MST signal for a given capillary. [36].

Y. Mao et al. / Talanta 132 (2015) 894–901896

above FITC stock solution with the volume ratio of 1:1. In the firstcompetitive binding study, the concentrations of FITC and BSA werefixed as 0.4 μmol L�1 and 1.2 μmol L�1, respectively. After FITC wasfully mixed with BSA (regarded as FITC-BSA), a serial dilution ofwarfarin (or ibuprofen) was added starting at 1 mmol L�1. In thesecond competitive binding study, the BSA was pre-saturated withwarfarin (or ibuprofen), and then the FITC with a fixed concentra-tion of 0.4 μmol L�1 was mixed with a series of warfarin (oribuprofen) saturated BSA. For the BSA aggregation investigation,the FITC labeled-BSA at the fixed concentration of 0.9 μmol L�1

were mixed with a series of unlabeled BSA at varying concentra-tions. All the solutions were filled in the standard glass capillariesafter the sample mixing and immediately measured by MST. Dataanalyses and curve fitting were carried out using the NanotemperAnalysis software. All the experimental parameters used by the MSTinstrument were fixed with a LED power of 20% and a laser powerof 80%.

2.6. Fluorescence spectroscopy experiments

In fluorescence measurements, BSA was introduced in anamount such that its concentration was always constant at0.02 mmol L�1 for each sample. FITC solution was pipetted intothese samples so as to obtain a series of quenching curves from0–0.14 mmol L�1, yielding 12 samples. The second series of solu-tions with the mixture of BSA and warfarin or ibuprofen (theirconcentration ratio was fixed as 1:1) was prepared as above andFITC was added to give different concentrations that ranged from0–0.036 mmol L�1, yielding 8 samples for the mixture of BSA andwarfarin. The third series FITC was added to yield a range ofconcentrations from 0–0.028 mmol L�1 and 7 samples for themixture of BSA and ibuprofen. All of these solutions were scannedon the fluorophotometer in the range of 300–450 nm with theexcitation wavelength of 280 nm at 298 K.

3. Results and discussion

3.1. Interaction between FITC and BSA by MST

The interaction between FITC and BSAwas investigated at roomtemperature and 37 1C with MST. During the experiment, the finalFITC concentration was fixed at 0.2 μmol L�1. By titrating FITCwith an increasing concentration of BSA, the binding curves wereobtained, which are given in Fig. 2. At the initial three points, dueto the low concentration of BSA compared to FITC, only a smallamount of FITC was bound to BSA, which resulted that the Fnorm,an indicator of thermophoretic property of binding partners, was

Scheme 1. Depiction of the competitive interaction between FITC-BSA and warfarin at room T or 37 1C.

Fig. 3. Competitive experiments by MST. The binding of warfarin to a constantamount of FITC-BSAwas quantified at room temperature (black dots) and 37 1C (reddots). Because of the binding in a competitive experiment with two ligandspresent, the fitting curves are just a guide to the eyes. The amplitude of Fnormsignal indicates the degree of competition. The final concentration of FITC and BSAwere fixed as 0.2 μmol L�1 and 0.6 μmol L�1, respectively, and warfarin withvarying concentrations was added. (For interpretation of the references to colorin this figure legend, the reader is referred to the web version of this article.)

Fig. 2. The binding curves of FITC with BSA obtained fromMST. FITC was titrated byBSA at room temperature (black dots) and 37 1C (red dots). The signal indicated thebinding behavior between BSA and FITC is a biphasic event. The FITC concentrationwas kept constant at 0.2 μmol L�1. The BSA concentration was ranging from1.0 mmol L�1. (For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.)

Y. Mao et al. / Talanta 132 (2015) 894–901 897

dominated by free FITC with its value about 860–870 units. As theBSA concentration increased, the Fnorm decreased first, indicating abinding process between FITC and BSA, and then recovered toalmost the same level at both room temperature and 37 1C, whichsuggests a biphasic binding pattern. At room temperature, FITChad a higher binding affinity for BSA with a Kd of 0.963 μmol L�1

(black) and a lower binding affinity with Kd of 23.8 μmol L�1 (red).At 37 1C, similar results were obtained with Kd of 0.783 μmol L�1

(blue) and 50.1 μmol L�1 (pink) for high and low binding affinities,respectively.

3.2. Competitive interaction study by MST

Sudlow's sites I and II are high affinity regions of BSA for avariety of molecules, such as dyes, drugs and small molecules. Tolocate the binding region of BSA upon binding with FITC, warfarinand ibuprofen were used as marker ligands for site I and II,respectively. A competitive strategy was applied to locate the FITCwhen binding to BSA. The final concentrations of FITC and BSAwere fixed at 0.2 μmol L�1 and 0.6 μmol L�1, respectively. AfterFITC was fully mixed with BSA (regarded as FITC-BSA), a serialdilution of warfarin was added starting at 1 mmol L�1. Thesesamples were measured by MST at both room temperature and37 1C, and the results are given in Fig. 3. At room temperature, the

value of Fnorm was constant at 860 units with the increasingconcentration of warfarin (0.01 μmol L�1 to 1 mmol L�1), whichvalue was consistent with that of FITC-BSA's at the same concen-tration in Fig. 2. This result implies that warfarin is not thecompetitor for the binding site between FITC and BSA, and FITCdoes not locate at site I. However, at 37 1C, the Fnorm value changedfrom about 800 units to 860 with the increasing concentration ofwarfarin. In Fig. 2, when the concentration of FITC and BSA were0.2 μmol L�1 and 0.6 μmol L�1, respectively, the Fnorm value was800 units at 37 1C. Therefore the Fnorm recovery from 800 to 860units in Fig. 3 was probably due to the replacement of FITC withwarfarin and the release of free FITC. This result suggests that FITCmight interact with BSA at site I to some extent at elevatedtemperatures. At the higher temperatures site I may open up topermit some FITC binding. This interaction process is depicted inScheme 1.

Subsequently, ibuprofen which has high affinity for the site II ofBSA was chosen as the competitor and added to the mixture ofFITC-BSA. The final concentration of FITC and BSA were still fixedas 0.2 μmol L�1 and 0.6 μmol L�1, respectively, and mixed thor-oughly. With the increasing addition of ibuprofen (0.01 μmol L�1

to 1.0 mmol L�1), the Fnorm signal increased slightly from about840 to 860 units at room temperature, which is indicative ofsubstitution of ibuprofen for FITC (Fig. 4). The released free FITCled to a recovery of Fnorm to 860 units. When the temperature wasincreased to 37 1C, similar results were obtained. The Fnorm valuestarted at 800 units, which is exactly the signal level for FITC-BSAthermophoresis, and finished at with about 860 units, in accor-dance with the signal level of free FITC. From these results, it isdeduced that ibuprofen has partly displaced FITC by binding at siteII. The competitive process is described in Scheme 2. Because thereplacement of FITC by ibuprofen happened at both room tem-perature and 37 1C, FITC is most likely more inclined to bind at siteII of BSA.

FITC is a frequently used organic dye to label proteins byforming covalent bonds with lysine or arginine in most commonlabeling method [32]. Usually in the most protocols, the labelingprocess requires several hours, but in this study, FITC only mixedwith BSA for several minutes. Whether this short incubation timewill lead to labeling process is unknown. Therefore it is necessaryto distinguish whether the interaction between FITC and BSA inthis study is a labeling process or weak interaction due toelectrostatic interaction, hydrophobic interaction, etc. Anothercompetitive strategy was used to make a distinction betweenthese two possible processes. Because warfarin and ibuprofenspecifically bind to sites I and II of BSA, respectively, the BSAwhich was saturated beforehand by warfarin or ibuprofen wassubjected to the FITC-BSA interaction study. When BSA is saturatedwith warfarin or ibuprofen, either site I or site II are completely

Scheme 2. Depiction of the competitive interaction between FITC-BSA and ibuprofen at room T or 37 1C.

Fig. 4. Competitive experiments by MST. The binding of ibuprofen to a constantamount of FITC-BSAwas quantified at room temperature (black dots) and 37 1C (reddots). Due to the presence of two ligands, the fitting curves are just a guide to theeyes. The final concentration of FITC and BSA were fixed as 0.2 μmol L�1 and0.6 μmol L�1, respectively, and ibuprofen with varying concentrations was added.(For interpretation of the references to color in this figure legend, the reader isreferred to the web version of this article.)

Y. Mao et al. / Talanta 132 (2015) 894–901898

occupied, which might lead to a weaker or unchanged interactionswith FITC depending on the different processes of binding.

A fluorescence quenching experiment was performed to deter-mine the minimum amount of warfarin and ibuprofen at whichBSA is saturated by achieving the maximum fluorescence quench-ing. According to the results, the fluorescence quenching effectwas maximized when the concentrations of warfarin and ibupro-fen were 50 and 300 times as much as that of BSA, respectively.

Subsequently, the final FITC concentration was fixed at0.2 μmol L�1 and mixed with a serial dilution of the mixture of BSAandwarfarin or ibuprofen (the concentration ratio was fixed as 1:50 forBSA and warfarin, and the ratio was fixed as 1:300 for BSA andibuprofen) starting at BSA concentration of 8.0 μmol L�1 at roomtemperature (Fig. 5). After data fitting, the Kd of FITC binding towarfarin-saturated BSA was 1.24 μmol L�1 and the Kd of FITC bindingto ibuprofen-saturated BSA could not be calculated accurately becausethe signals never reached a stable value. The difference between thesetwo curves excluded the possibility of the FITC labeling process,because if FITC had formed covalent interactions with BSA, thedissociation constant would remain constant regardless of the presenceof warfarin or ibuprofen. However, the Kd of FITC binding to ibuprofen-saturated BSA could not be obtained and differed from the Kd of FITC

binding to BSA, and thus FITC interacts with BSA by a weak interactioninstead of a labeling process. The Kd of FITC binding to warfarin-saturated BSA is approximately equal to that of FITC binding to freeBSA, which demonstrates that thermophoretic movement is notaffected by excess amount of warfarin. This result indicates that FITCis more inclined to have a higher affinity for site II of BSA, which isdisplayed in Scheme 3. When FITC added to the ibuprofen-saturatedBSA, because site II was occupied by ibuprofen, a lower binding affinitywas achieved, confirming that FITC binds biphasically, FITC tends tointeract with site II of BSA to a large extent, and with site I to a lesserdegree.

3.3. Property of BSA in solution

In Fig. 2, a biphasic binding pattern was observed, whichindicates two forms of BSA binding to FITC. To elucidate thereason for the increase of Fnorm with respect to BSA concentration,a serial dilution of BSA was added into FITC-labeled BSA. The FITC-labeled BSA was fixed at 0.9 μmol L�1. With the increasingaddition of free BSA, a binding curve was obtained with the Kd

of 2.01 μmol L�1, which indicates that BSA tends to aggregate atthis concentration (Fig. 6). The value of Kd is almost in line with theconcentration (about 2.0–4.0 μmol L�1) corresponding to the

Fig. 6. MST quantified aggregation of BSA. A constant amount of labeled BSA–FITC(9.0 μmol L�1) was titrated by unlabeled BSA. The value of Kd was corresponding tothe lowest value of Fnorm from the interaction between FITC and BSA.

Scheme 3. Depiction of FITC interacting with BSA in the presence of site markers-saturated.

Fig. 5. The binding curves of FITC with warfarin-saturated BSA or ibuprofen-saturated BSA. The signal of black dots showed a constant amount of FITC(0.2 μmol L�1) was titrated by warfarin-saturated BSA (the concentration ratio ofBSA and warfarin was 1:50). The signal of red dots showed a constant amount ofFITC (0.2 μmol L�1) was titrated by ibuprofen-saturated BSA (the concentrationratio of BSA and ibuprofen is 1:300). (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of this article.)

Y. Mao et al. / Talanta 132 (2015) 894–901 899

lowest value of Fnorm in Fig. 2, confirming that FITC binds to theaggregate of BSA which results in a second binding process.

3.4. Fluorescence measurements

MST is a new method for biomolecular interaction investiga-tion, and thus conventional fluorescence spectroscopy methodwas applied to verify the results obtained from MST. Warfarin andibuprofen were still selected respectively as the marker of site I orsite II for the competitive study.

BSA shows intrinsic fluorescence mainly due to the two trypto-phan residues [33], which are strongly affected by the interactionbetween BSA and molecules. When the increasing amounts of FITCwere added to a BSA solution of constant concentration, thefluorescence of tryptophan at 340 nm decreased gradually withoutobvious shift in emission wavelength (As can be seen in Fig. 7). Thefluorescence intensity decrease is probably attributed to dynamicquenching, static quenching or both of the two. To clarify themechanism, the quenching process was first assessed by the Stern-Volmer equation [34] assuming a dynamic quenching:

F0=F ¼ 1þKqτ0½Q � ¼ 1þKSV ½Q �

In the above equation, F0 and F are the fluorescence intensitiesof the protein before and after the addition of quencher (in thiscase, FITC), respectively; Kq is the quenching rate constant of thebiomolecule; τ0 is the average lifetime of the protein withoutthe quencher, and generally taken as 10�8 s; [Q] is the concentra-tion of the quencher, and KSV is the dynamic quenching constant.The Stern–Volmer plots for the interaction of FITC and BSA werecalculated to be in accordance with linear model. The value ofKSV was 3.1�105 L mol�1 (with a correlation coefficient r of0.9987) and the corresponding Kq was calculated as about 3.1�1013 L mol�1 s�1, which was much larger than the maximumscatter collision quenching constant (2.0�1010 L mol�1 s�1) forbiomolecules. Therefore, the interaction between FITC and BSA ispossibly a process of static quenching rather than dynamicquenching.

Due to the deduction that the binding of FITC to BSA is a staticfluorescence quenching process, which results in the formation ofnon-fluorescent compound, the fluorescence intensity could bedescribed by the Scatchard equation [35]

lg ½ðF0�FÞ=F� ¼ lg Kaþn lg ½Q �

Fig. 8. (A) Fluorescence emission spectra of BSA-warfarin (0.02 mmol L�1) (their concentration ratio was fixed as 1:1) in the presence of FITC with different concentrations(from a to h: 0.000, 0.012, 0.016, 0.020, 0.024, 0.028, 0.032 and 0.036 mmol L�1). (B) Fluorescence emission spectra of BSA-ibuprofen (0.02 mmol L�1) (their concentrationratio was fixed as 1:1) in the presence of FITC with different concentrations (from a to h: 0.000, 0.004, 0.008, 0.012, 0.016, 0.020 and 0.024 mmol L�1).λex¼280 nm. Inset: thelinear relationship for quenching.

Fig. 7. Fluorescence emission spectra of BSA (0.02 mmol L�1) in the presence ofFITC with different concentrations (0–0.14 mmol L�1). λex¼280 nm. Inset: thelinear relationship for quenching BSA by FITC.

Y. Mao et al. / Talanta 132 (2015) 894–901900

for which Ka is the binding constant, implicating the degree ofinteraction between BSA and FITC, and n is the number of bindingsites. Depending on the linear relationship between lg[(F0�F)/F]and lg[Q], Ka was calculated to be 3.47�106 L mol�1. The recipro-cal of this Ka value (2.88�10�7 mol L�1) is on the same order ofmagnitude as the dissociation constant Kd obtained by MST, whichsuggests that MST provides reliable results. For comparison, theinfluence of warfarin or ibuprofen on the binding of FITC to BSAwas investigated by titrating the pre-mixed 1:1 BSA-warfarin (oribuprofen) solution with a series concentration of FITC (as given inFig. 8A and B). The increasing amount of FITC produced a gradualdecrease in fluorescence intensity of BSA-warfarin (or BSA-ibu-profen) mixture and the corresponding Ka was calculated, whichare listed in Table 1. The binding constants changed from 3.47�106 L mol�1 to 2.09�106 L mol�1 and 0.93�106 L mol�1 in thepresence of warfarin and ibuprofen, respectively. Accordingly, thedecrease of Ka in the presence of ibuprofen was much larger thanthat in the presence of warfarin, which also indicates that FITCmainly tends to bind with the site II of BSA, and with the site I ofBSA to a smaller extent. These results are consistent with thosefrom MST.

4. Conclusions

In this study, a novel approach microscale thermophoresis wasapplied to investigate the interaction between BSA and a smallorganic dye FITC. FITC exhibits a strong binding affinity for BSA. FITCis mainly prone to interacting with the hydrophobic pocket of site IIof BSA, while it also binds to site I of BSA to a lesser degree. Theseresults from MST were in accordance with those from fluorescencespectroscopy, indicating the feasibility and reliability of MST in theapplication of protein–small molecule interaction. In addition, MSTalso demonstrated that FITC not only binds to BSA, but also binds toBSA aggregates, providing some detailed information about the BSAaggregation which was not obtained by fluorescence spectroscopy.Therefore MST is a powerful and reliable approach for protein–smallmolecule interaction. Combined with other advantages of MST, suchas the low sample consumption which only needs several micro-litres and nano molarity level, the simplicity of mixing, the fastmeasurement, the diversity of analytical solution especially in nativeenvironment and so on, MST is notably suitable in the applicationinvolved precious and expensive sample analysis.

Acknowledgments

This work was financially supported by the National NaturalScience Foundation of China (21002093) and the national science andtechnology support program project funds (No. 2012BAD37B04).

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Table 1Binding constants of FITC-BSA system in the absence and presence of site markersat 298 K.

Site marker Scatchard equation r Ka/106 L mol�1

Without markers Y¼(1.36)Xþ6.55 0.9957 3.47Warfarin Y¼1.32Xþ6.32 0.9993 2.09Ibuprofen Y¼1.23Xþ5.97 0.9990 0.93

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