Thermochimica Acta 575 (2014) 291299Contents lists available at ScienceDirectThermochimicaActaj our nal homepage: www. el sevi er . com/ l ocat e/ t caSynthesis,thermodynamicpropertiesandBSAinteractionofanewValenShiffbasederivedfromo-vanillinandtrimethoprimXuLia,Jian-HongJianga,Sheng-XiongXiaoa,Hui-WenGub,, Chuan-HuaLia,Li-JuanYea,XiaLia,Du-GuiHea,Fei-HongYaoa,Qiang-GuoLia,aHunan Provincial Key Laboratory of Xiangnan Rare-Precious Metals Compounds and Applications, Department of Chemistry and Life Science, XiangnanUniversity, Chenzhou 423000, Hunan Province, PR ChinabState Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082,HunanProvince, PR Chinaart icleinfoArticle history:Received 2 September 2013Received in revised form16 November 2013Accepted 19 November 2013Available online 27 November 2013Dedicated to Professor Qiang-Guo Li on theoccasion of his 60th birthday.Keywords:o-VanillinTrimethoprimValen Shiff baseStandard molar enthalpy of dissolutionStandard molar enthalpy of formationBovine serumalbuminabstractAnewValenShiffbase(C22H24N4O5)was synthesizedusingequivalentmolesofo-vanillinandtrimetho-prim.At 298.15K,thestandardmolarenthalpyofformationofthenewcompoundwas estimatedtobe

fH

m [C22H24N4O5(s),298.15K]=(696.921.67)kJmol1by microcalorimetry.In particular,theinter-actionbetweentheShiffbaseandbovineserumalbumin(BSA)has beeninvestigated.ItwasprovedthattheuorescencequenchingofBSAby Shiffbaseisa resultoftheformationofaShiffbase-BSAcomplex.QuenchingconstantsweredeterminedusingtheSternsVolmerequationtoprovideameasurementofthebindingsitebetweenShiffbaseandBSA.ThethermodynamicparametersG,H,andSofthesys-tematdifferenttemperatureswerecalculated.Whatismore,thedistancer betweendonor(Trp.213)andacceptor(Shiffbase)wasobtained.Finally,synchronousuorescencespectroscopydatahassuggestedthe associationbetweenShiffbaseandBSAchangedthemolecularconformationofBSA. 2013 Elsevier B.V. All rights reserved.1. IntroductionAs one of the most important compound categories in coor-dination chemistry, Shiff bases and their corresponding metalcoordination complexes have been widely applied in many eldssuch as biological medicine, catalytic synthesis, analytical chem-istry, anticorrosion, light-induced discoloration, and so on [1,2].In recent years, the studies on Shiff base compounds have beenfocused on their biological activities (antibacterial and antitumoractivities), catalytic properties, etc. Researchers have designed andsynthesized many Shiff bases and their metal coordination com-plexes, which proved to possess good antibacterial activities [38],but there is still a long way to go before these kinds of compoundscan be used for clinical applications. Considering their potentialCorresponding author. Tel.: +86 735 2653353; fax: +86 735 2653353.Corresponding author. Tel.: +86 182 73153402; fax: +86 182 73153402.E-mail addresses: gruyclewee@hnu.edu.cn (H.-W. Gu), liqiangguo@163.com(Q.-G. Li).applicationvalues inpharmaceutical chemistry, it is very necessaryto conduct a further study on these kinds of Shiff base compounds.Valen Shiff bases are such kind of compounds derived fromthecondensation reaction of o-vanillin and amine, whose antibacte-rial activities have not been studied enough for the researchingabout Valen Shiff bases and their metal coordination complexesmainly concentrates on the aspects of catalytic agents, antioxi-dants, and luminescent materials [911]. In fact, it is reported thatthe antibacterial activity of o-vanillin is better than that of salicy-laldehyde [12,13]. Likewise, sometimes the antibacterial activitiesof some Valen Schiff bases maybe also superior to those of salic-ylaldehyde Schiff bases [14]. In addition, since Valen Shiff basescan provide the atomof oxygen which has lone pair electrons andthus possess good coordination abilities to some hard acids suchas rare metal ions, researchers can obtain various kinds of coor-dination complexes derived from them, which could be used asligands. Accordingly, designing and synthesizing different kinds ofValen Shiff bases and their metal coordination complexes with var-ious structures can lay a foundation for the further development ofhigh efcient, low toxic Valen Shiff base coordination complexeswhich could be used as drug candidates. And it can also benet the0040-6031/$ see front matter 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.tca.2013.11.015292 X. Li et al. / Thermochimica Acta 575 (2014) 291299research of structureactivities relationship of these kinds of ValenShiff base compounds.Trimethoprim (5-(3,4,5-trimethoxybenzyl)-2,4-pyrimidinedia-mine, CAS No. 738-70-5, see Fig. S1) is a bacteriostatic antibioticmainly usedinthe prophylaxis andtreatment of urinary tract infec-tions. The molecular formula of trimethoprim is C14H18N4O3. Itbelongs to the class of chemotherapeutic agents knownas dihydro-folate reductase inhibitors. Trimethoprimis an antibacterial agentandits curativeeffect increases whenusedincombinationwithsul-fonamide drugs such as sulfadiazine or sulfadoxine. It is applicableto many diseases, such as respiratory tract infection, senile chronicbronchitis, bacillary dysentery, urinary tract infection, enteritis,typhia and malaria, etc. [15].As can be known fromthe structure of trimethoprim, there aretwo amino groups in its molecular. If trimethoprim reacts withdifferent amounts of o-vanillin, single or double Valen Shiff basecould be obtained. In this paper, a new single Valen Shiff basewas synthetized using equivalent moles of o-vanillin (C8H8O3)and trimethoprim (C14H18N4O3), whose composition and struc-ture were characterized by element analysis, molar conductance,UVvis spectroscopy, IR spectroscopy, and NMRspectroscopy. Thethermodynamic properties of the new synthetic Valen Shiff basewere investigated using an advanced solution-reaction isoperibolmicrocalorimeter. Inparticular, wealsoinvestigatedtheinteractionbetween BSA and this newsynthetic Shiff base with the assistanceof uorescence analysis. Webelieve that such research maybenetthe development and application of Valen Shiff bases in the eld ofmedicine, bioinorganic chemistry, and molecular biology.2. Experimental details2.1. Reagents and instrumentso-Vanillin was purchased from Beishun Chemical Technol-ogy Co., Ltd. (Beijing, China). Trimethoprim was obtained fromSinopharmChemical Reagent Co., Ltd. (Beijing, China). Their labeledmass fraction purities were 99.0% and 98.0%, respectively. Afterpurication, the actual mass fraction purities of twosamples weredetermined to be greater than 99.5% by HPLC. KCl (calorimetric pri-mary standard, mass fraction purity greater than 99.99%) was driedin a vacuum oven at 135C for 6h before use. BSA was obtainedfrom Shanghai Boao Biological Technology Co., Ltd. (Shanghai,China). All other chemicals used were analytical or biological gradeand used without further purication. The water used wastriplydistilled.The elemental analysis of C, H and N was performed with anelemental analyzer (Perkin-Elmer 2400 CHN, USA). FTIR spectra(4000400cm1) were recorded on a Fourier transform IR spec-trometer with a KBr pellet (Avatar360, Nicolet, Madison, USA).Ultraviolet spectra were obtained on an UV-vis spectrophotometer(U-3010, Hitachi, Tokyo, Japan).1H NMRand13C NMRmea-surements were run on a 400MHzNMRspectrometer (BruckerAvance, Switzerland) using trimethyl silicane (TMS) as the inter-nal reference standard. The uorescence spectra were recordedon an F-4600 uorescence spectrophotometer (Hitachi, Tokyo,Japan). The molar conductance was determined by a digital Abberefractometer (WAY-IS, Shanghai Precision & Scientic InstrumentCo., Ltd., Shanghai, China). The dissolution enthalpies were mea-suredby anSCR-100 solution-reactionisoperibol microcalorimeter(constructed by the Thermochemical Laboratory of Wuhan Univer-sity, China). The principle and structure of the microcalorimeterhave been detailed in the literatures [16,17]. The volume of thereaction vessel was 100cm3, and the precision of the test tem-perature and control temperature were 0.001K and 0.0001K,respectively.2.2. Synthesis of the Valen Shiff base derived fromo-vanillin andtrimethoprimA 250mL three-necked ask equipped with a condenser pipeand a 100mLseparating funnel wasused as the reactor. 2.9032gtrimethoprim(10mmol) was dissolvedinthe 250mLthree-neckedask with 70mLmethanol, then the solution was put into anoil bath to keep a constant temperature of 55C and stirredusing a magnetic stirrer until the solid wasdissolved completely.1.5214g (10mmol) o-vanillin wasdissolved in 50mLmethanoland then transferred into a 100mLseparating funnel. After that,the methanol solution of o-vanillin was added dropwise into themethanol solution of trimethoprim. As the reaction progressed, thecolor of the mixture changed from colorless to yellow, and nallybecame dark red. The reactionwascontinuedfor 6h, resulting innoprecipitationgenerated. Subsequently, the solvent was preliminaryremoved via vacuum distillation, and the remaining solution wasdriedina vacuumdesiccator at 55Cto obtainplenty of yellowpre-cipitate. The product was washed alternately with distilled waterand ethanol, ltered and dried under vacuum until the weight ofthe powders remained constant, with a yield of 65%.Element analysis, molar conductance, UVvis spectroscopy, IRspectroscopy, and NMRspectroscopy were employed to char-acterize the composition and structure of the Shiff base. Theresults (details in the Supplementary data) indicated that thesynthetic Shiff base has a composition of C22H24N4O5, and itsstructure is shown in the following Fig. 1. According to the nomen-clature of IUPAC, the IUPAC name of this new synthetic ValenShiff base is 2-{[4-amino-5-(3,4,5-trimethoxy-benzyl)-pyrimidin-2-ylimino]-methyl}-6-methoxy-phenol.2.3. The solution-reaction isoperibol microcalorimeter andcalibrationThe solution-reaction isoperibol microcalorimeter with a con-stant temperature environment wasconstructed and calibratedaccording to the published literatures [16,17]. In the calori-metric experiments, the temperature was 298.15K, the currentwas 21.813mA,and the resistance of the heater was1212.3.The calibration of the calorimeter wascarried out by measuringthe dissolution enthalpies of KCl (calorimetric primary standard)in triply distilled water and Tris(hydroxymethyl)aminomethane(THAM, NBS 742a) in 0.0001molcm3HCl at 298.15K. The meandissolution enthalpies were (17,59717) J mol1for KCl and(29,77616) J mol1for THAM, inagreement withpublisheddata(17,5369) J mol1for KCl [17] and (29,76631.5) J mol1forTHAM [18]. The eventual errors of the experimental results werewithin 0.5% compared with the recommended reference data,which suggests that the microcalorimeter wasfeasible.2.4. Determination of dissolution enthalpies2.4.1. Thermochemical cycle of the synthetic reaction of Shiff baseAlthough the thermal effect of the solid-state synthetic reac-tion wasdifcult to determine, it wasfeasible to deduce theenthalpies of formation fromthe dissolution enthalpies measuredwhen the samples were dissolved in the calorimetric solvent. Inthis work, a convincing thermochemical cycle based on Hesss lawwas designed, as shown in Fig. 2.The UV spectra and refractive indexes of the nal solution ofthe reactants and the nal solution of the products can be usedto determine whether they have the same thermodynamic state.In the present experiments, we determined the UV spectra andrefractive indexes of solutions B and D in Fig. 2. The experimentalresults suggested that the twosolutions have similar UVspectra(see Fig. 3) and equal refractive indexes (298.15K=1.3972), whichX. Li et al. / Thermochimica Acta 575 (2014) 291299 293Fig. 1. Chemical structure (A) and 3D structure (B) of the synthetic Valen Shiff base.C8H8O3(s) C14H18N4O3(s)H2O(l)+Solution B+ Calorimetric Solvent SSolution DSolution ASolution Cr HmC22H24N4O5(s) ++ Calorimetric Solvent Ss Hm(b)s Hm(c)s Hm(d)s Hm(a)Fig. 2. Thermochemical cycle of the synthetic reaction of Valen Shiff base.demonstrates that they have the same thermodynamic state andthat the designed thermochemical cycle of the synthetic reactionwas reliable.2.4.2. Choice of calorimetric solventIt is veryimportant tochoosea solvent that candissolveall of thereactants and products rapidly and completely. Research indicatedthat the relevant substances in the synthetic reaction are highlysoluble in the mixed solvent of dimethylformamide (DMF), ace-tonitrile(ACN) andacetic acid(HAC). Bymixingthesethreesolventsuniformlyat different ratios andcontinuallytestingthe solubilityofthe samples, the best solubility was foundwhenthe volume ratio ofthe three solvents was VDMF/VACN/VHAC=1:1:1. Consequently, thismixture was chosen as the optimal calorimetric solvent S.800 700 600 500 400 300 200 100012345absorbance/nm Solution B Solution DFig. 3. UVvis spectra of the nal dissolution state of the reactants and products.2.4.3. Determination of dissolution enthalpies of all the chemicalsin the synthetic reactionThe experimental conditions of determination of dissolutionenthalpies were the same as those of the calorimeter calibrationmentioned in Section 2.3.Thoroughly dried samples were ground completely in an agatemortar, and then exactly 0.5mmolof sample wasplaced in thesample container of the microcalorimeter. The calorimetric solventS (100.00mL)had been added to the reaction vessel in advance.When the calorimeter wasadjusted to a constant temperature of(298.1500.001) K, the samples were added to the reaction vessel,and then their dissolution enthalpies were measured. The resultsare listed in Table 1 based on ve parallel measurements.2.5. The interaction between BSA and the synthetic Valen ShiffbaseThe stock solution of BSA with a concentration of 5gL1wasprepared by diluting appropriate BSAin 0.05mol L1NaCl solution,andthenstoredat 4Cina refrigerator. The synthetic Shiff base wasdissolved in a mixture solvent (VDMSO/VH2O = 1 : 4) with a nalconcentration of 1.0104molL1. TrisHCl buffer was preparedwith5mmol L1Tris and50mmolL1NaCl andadjustedtopH=7.4with hydrochloric acid.2.5.1. Fluorescence spectraDilute the stock solution of BSA to prepare the working solu-tion with a nal concentration of 0.5gL1in 0.05molL1NaClsolution. 11 aliquots of 2.68mLBSA working solution were pipet-ted into 10mLasks. Different amounts of the synthetic Shiff base(cShiff/cBSA=010) were added into the aforementioned asks con-taining equal amount of BSA (2.0106molL1), and dilute themixture solution with TrisHCl buffer to 10mL. The widths of boththe excitation slit and the emission slit were set to 3nm. Under theexcitation wavelength of 280nm,the emission spectra from300 to500nmwere recorded. To investigate the uorescence quenchingof BSA by the Shiff base, the uorescence measurements were per-formed at four different temperatures (T =298.15, 302.15, 306.15,and 310.15K).2.5.2. Synchronous uorescence spectroscopyThe test solutions were prepared as described in Section 2.5.1.The excitation and emission monochromator run at the same time.The relationshipof the synchronous emissionwavelengthandexci-tationwavelengthcanbe expressedas em=ex+. Synchronousuorescence spectra were recorded when the value of was setto =15nmand =60nm,respectively.294 X. Li et al. / Thermochimica Acta 575 (2014) 291299Table 1The dissolution enthalpies of [C8H8O3 (s)], [C14H18N4O3 (s)] and [C22H24N4O5 (s)] in the calorimetric solvent S at 298.15K.System No. m(g) t (s) s H

m(kJ mol1)C8H8O3(s)in solvent S1 0.0761 20.50 22.695020.0760 18.70 22.773330.0761 17.96 22.913340.0762 17.87 22.790450.0762 16.91 23.5033s H

m[C8H8O3 (s),298.15K] =(22.940.33a) kJ mol1C14H18N4O3(s)in solvent S1 0.1452 9.19 9.552220.1451 8.32 10.15563 0.1452 7.75 8.626540.1453 6.78 9.137350.1452 7.00 9.4596sH

m[C14H18N3O3 (s),298.15K] = (9.39 0.56) kJ mol1C22H24N4O5(s)in solvent S1 0.2082 11.56 14.55982 0.2082 16.78 14.325330.2082 11.74 14.226940.2083 11.74 14.033250.2081 11.52 14.1894sH

m[C22H24N4O5 (s),298.15K] = (14.27 0.19) kJ mol1m: Mass of the sample; t: heating period of electrical calibration.aUncertainty was estimated as twice the standard deviation of the mean of the results.2.5.3. UVvis absorbance spectraThe UVvis absorbance spectrum of the synthetic Shiff base(2.0106molL1) was measured using Trisbuffer as a reference.3. Results and discussion3.1. Results of calorimetric experiment and data treatment3.1.1. Results of calorimetric experimentThe dissolution enthalpies of [C8H8O3 (s)], [C14H18N4O3 (s)] and[C22H24N4O5 (s)] in the calorimetric solvent S at 298.15K are listedin Table 1.3.1.2. Data treatment3.1.2.1. Estimation of sH

m(d). sH

m(d) denotes the dissolutionenthalpy of 0.5mmolH2Oin solution C (as shown in Fig. 2). Accord-ing to literature [19], when the concentration of solute in a solutionis extremely low, this solution can be regarded as pure solvent. Theadditionof small amount of other pure solvent into the solutionhaslittle effect on the whole system, and in this case the dissolution ofthe small amount of addition solvent in the extremely dilute solu-tion can be ignored. In the current experiment, the concentrationof solution C is 5.0106molL1, and the amount of the additionH2O into solution C is 0.5mmol. Accordingly, we can assume thatthe additionof 0.5mmolH2OintosolutionCwouldhave little effecton the whole system, and thus here sH

m(d) is ignored.3.1.2.2. Standard molar reaction enthalpy of the synthetic reaction.According to Hesss law, the standard molar reaction enthalpy ofthe synthetic reaction can be expressed as follows:

rH

m = sH

m(a) +sH

m(b) sH

m(c) sH

m(d)= sH

m[C8H8O3(s), 298.15K]+sH

m[C14H18N4O3(s),298.15K]sH

m[C22H24N4O5(s),298.15K]sH

m[H2O(l), 298.15K]In the above equation, sH

mrepresents the dissolutionenthalpies of the reactants and products of the synthetic reactionin the calorimetric solvent S at 298.15K, which were determinedusing an advanced solution-reaction isoperibol microcalorimeteras described detailed in Section 2.4.3. Substituting the correspond-ing data into the equation gives

rH

m = (22.94 9.39 +14.27 0)

0.332+0.562+0.192+02= (27.82 0.68)kJ mol13.1.2.3. Calculation of fH

m[C22H24N4O5(s), 298.15K]. Accordingto Hesss lawand the principles of thermodynamics, we have

rH

m = fH

m[C22H24N4O5(s),298.15K]+fH

m[H2O(l),298.15K]fH

m[C8H8O3(s), 298.15K]fH

m[C14H18N4O3(s), 298.15K]According to refs [20,21]

fH

m[C8H8O3(s), 298.15 K] = (487.23 0.96) kJ mol1

fH

m[C14H18N4O3(s), 298.15K] = (523.34 1.19)kJ mol1

fH

m[H2O(l), 298.15K] = (285.830 0.040) kJ mol1so that

fH

m[C22H24N4O5(s), 298.15K]= rH

m fH

m[H2O(l),298.15K]+fH

m[C8H8O3(s), 298.15K]+fH

m[C14H18N4O3(s), 298.15K]= (27.82 487.23 523.34 +285.830)

0.682+0.962+1.192+0.0402= (696.92 1.67)kJ mol1X. Li et al. / Thermochimica Acta 575 (2014) 291299 295300 350 400 450 500050100150200250300Fluorescence intensity (a.u.)/nmFig. 4. The quenching effect of Shiff base on BSA uorescence inten-sity. ex/em =280nm/340nm,cBSA =2106molL1. The concentration ratio(cSchiff/cBSA) fromA to K is 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, respectively.3.2. The interaction between BSA and Shiff base3.2.1. Effect of Shiff base on BSA uorescenceFluorescence quenching refers to any process which decreasesthe uorescence intensity of a sample such as excited state reac-tions, energy transfers, ground-state complexes formation andcollisional process [22]. BSA has three intrinsic uorophores: tryp-tophan, tyrosine and phenylalanine that can be quenched. In fact,as Sulkowska [23] said, because phenylalanine has a very lowquantum yield and the uorescence of tyrosine is almost totallyquenched if it is ionized, or near an amino group, a carboxyl group,or a tryptophan, the intrinsic uorescence of BSA is almost con-tributed by tryptophan alone. Here Shiff base is a quencher whichcanquenchthe uorescence intensityof BSAwhenbondtoBSA. Wemeasured the uorescence quenching emission spectra of BSA atvarious concentrations of Shiff base under physiological condition.FromFig. 4, we cansee clearlythat BSAhada stronguorescenceemission band at 340nm by xing the excitation wavelength at280nm,which is mainly contributed by the tryptophan in BSA;while Shiff base had no intrinsic uorescence due to there is no bigconjugate systemof double bond in its molecular. The uorescenceintensity of BSAdecreased remarkably and the maximumemissionwavelength remained a constant with the increasing of Shiff baseconcentration, indicating that interactions between Shiff base andBSA occurred.3.2.2. The uorescence quenching mechanismThe different mechanisms of quenching are usually classiedas dynamic quenching and static quenching. In static quenching,the quencher forms a non-uorescent complex with the uo-rophore of the macromolecule, whereas in dynamic quenching thequencher binds to the uorophore during the life time of excitedstate, which induces charge transfer from the uorophore to thequencher and consequently the uorophore turns to the groundstate without emitting a photon [24]. Experimentally, dynamic andstatic quenchingcanbedistinguishedbytheir differingdependenceon temperature and viscosity. Since higher temperature resultsin larger diffusion coefcients, the dynamic quenching constantsare expected to increase with increasing temperature. In contrast,increased temperature is likely to result in decreased stabilityof complexes, and thus lower values of the static quenchingconstants [25]. Generally, quantitative analysis of the quenchingphenomenonis performedvia applying the SternVolmer equationto the uorescence quenching data:F0F= 1 +KSV[Q] = 1 +kq 0[Q] (1)orF0FF=KSV[Q] (2)where F0and F are the uorescence intensities in the absenceand in the presence of the quencher at concentration [Q], respec-tively; 0 is the average lifetime of the uorophore in the absenceof quencher; kq is the bimolecular quenching constant; KSV is theSternVolmer quenching constant (Kd) or association constant (Ka)in dynamic or static quenching, respectively.To clarify the uorescence quenching mechanism of BSAby Shiff base, it wasrst assumed that the interactionproceeds via a dynamic way. The temperature-dependentuorescence quenching of BSA by Shiff base wasthen car-ried out. The SternVolmer plots at different temperatureswere shown in Fig. 5A. From the experimental data, the cor-responding dynamic quenching constants for the interactionbetween Shiff base and BSA were KSV=1.06106L mol1(298.15K, R=0.9996), KSV=1.02106L mol1(302.15K,R=0.9985), KSV=0.94106L mol1(306.15K, R=0.9987), andKSV=0.91106L mol1(310.15K, R=0.9977), respectively.Because the uorescence lifetime of the biopolymer is 108s[2628], the quenching constants kq at 298.15, 302.15, 306.15 and310.15Kwere calculated to be 1.061014, 1.021014, 0.941014,and 0.911014L mol1s1, respectively.According to the literatures [28,29], for dynamic quenching,the maximum scattering collision-quenching constant of variousquenchers with the biopolymer is 2.01010L mol1s1, and theKSV increases with increasing temperature. Considering that in ourexperiment the rate constant of the protein quenching procedureinitiated by Shiff base is much greater than 2.01010L mol1s1and that the KSV decreased with increasing temperature, it can beconcluded that the quenching is not initiated by dynamic quench-ing, but probably by static quenching resulting fromthe formationof Shiff base-BSAcomplex. Consequently, the quenching data couldbe analyzed according to the following LineweaverBurk equation[30]:1F0F =1F0+1KLB F0[Q](3)In the present case, F0and F are the uorescence intensitiesin the absence and in the presence of the quencher at concentra-tion [Q], respectively. KLB is the effective static quenching constantfor the accessible uorophores, which is analogous to associativebinding constant for the quencheracceptor system.The LineweaverBurk plots are shown in Fig. 5B, and thecorresponding static quenching constants KLB at four different tem-peratures are listed in Table 2. As can be seen from Table 2, thedecreasing trend of static quenching constant (KLB) with increasingtemperature wasin accordance with KSVs dependence on temper-ature as mentioned above, which in turn is an indication for staticquenching.3.2.3. Thermodynamic parameters and nature of binding forcesThe interaction forces between a drug and a biomolecule mayinvolve hydrophobic forces, electrostatic interactions, van derWaals interactions, hydrogen bonds, etc. According to the data ofenthalpy change (H) andentropy change (S), the model of inter-action between a drug and a biomolecule can be concluded [31]:296 X. Li et al. / Thermochimica Acta 575 (2014) 2912991.0 0.8 0.6 0.4 0.2 0.00.00.20.40.60.81.01.2 B:T=298.15K;Slope=1.0622;R=0.9996 C:T=302.15K;Slope=1.0194;R=0.9985 D:T=306.15K;Slope=0.9414;R=0.9987 E:T=310.15K;Slope=0.9133;R=0.9977(F0-F)/FCq106/(molL-1)(A)10 8 6 4 2 00.0050.0100.0150.0200.0250.0300.0350.0400.0450.050 B:T=298.15K, Slope=0.00277, R=0.9976 C:T=302.15K, Slope=0.00301, R=0.9875 D:T=306.15K, Slope=0.00379, R=0.9962 E:T=310.15K, Slope=0.00425, R=0.99601/(F-F)01/(C10q6/(molL-1))(B)Fig. 5. SternVolmer plots (A) and LineweaverBurk plots (B) for the uorescence quenching of BSA by Shiff base at different temperatures (298.15, 302.15, 306.15 and310.15K).Table 2Relative thermodynamic parameters of the systemof Shiff base-BSA.Temperature (K) KLB (L mol1) H

(kJ mol1) G

(kJ mol1) S

(J mol1K1)298.15 1.31710622.99734.929 40.020302.151.24110635.248 40.546306.15 1.01310635.198 39.853310.159.19810535.409 40.019(1) H>0 and S>0, hydrophobic forces.(2) H