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Fluorometric investigation on the interaction of oleanolic acid
with bovine rum albumin
Zhengjun Cheng,Yuntao Zhang
*
Institute of Applied Chemistry,China West Normal University,Nanchong Sichuan 637002,China Received 20May 2007;received in revid form 8August 2007;accepted 15August 2007
Available online 29August 2007
Abstract
The interactions between oleanolic acid and bovine rum albumin (BSA)have been studied by fluorescence,circular dichroism (CD),UV–vis absorption and Fourier transform infrared spectroscopy (FTIR)under physiological conditions.Spectroscopic analysis of the emission quenching at different t
emperatures has revealed that the quenching mechanism of bovine rum albumin by oleanolic acid is static quenching mechanism.The binding sites number n and binding constants K are obtained at various temperatures.The distance r between oleanolic acid and the protein is evaluated according to the theory of Forster energy transfer.The results by FTIR,CD and UV–vis absorption spectra experiment indicate that the condary structures of protein have been perturbed in the prence of oleanolic acid.The thermodynamic parameters D H 0,D G 0,and D S 0are calculated according to van’t Hoffequation,which indicates that the hydro-gen bonds and van der-waals are the intermolecular forces stabilizing the complex.Molecular modeling studies the interaction BSA with oleanolic acid.
Ó2007Elvier B.V.All rights rerved.
Keywords:Bovine rum albumin;Oleanolic acid;Fluorescence spectroscopy;Circular dichroism spectroscopy;Fourier transform infrared spectroscopy
1.Introduction
Oleanolic acid (3b -hydroxy-olea-12-en-28-oic acid,OA,Fig.1)is one of the best known bioactive pentacyclic trit-erpenoids,which exits widely in almost 190species of 60branches of food,medicinal h
erbs,and other plants as the form of free acid or aglycones for triterpenoid saponins [1].It has been reported to have numerous pharmacologi-cal activities including anti-inflammatory,anti-cancer,anti-HIV [2],hepato-protection,analgesia,cardiotonic,gluco lowering [3],inhibitory activity of tumor growth and the therapeutic utility of protecting bone marrow from the damages of radiation [4],healing of chronic gastric lesions in rats [5],Metallothionein (MT)[6],protection against carbon tetrachloride-induced and cadmium hepato-toxicity [7],antitubercular potential against mycobacte-
rium tuberculosis [8].In recent years,it has been found that it had exhibited cytotoxic activity toward many cancer cell lines in culture [1].
Serum albumins are abundant with proteins in blood plasma,accounting for about 60%of the total protein cor-responding to a concentration of 42g dm À3and provide 80%of the osmotic pressure of blood [9,10].As a con-quence,albumins have been ud as model proteins in v-eral biophysical,biochemical,and physicochemical studies for many years [11].Bovine rum albumin (BSA)is a very interesting biophysical and biochemical system and has been quite well studied in the past 40years [12].BSA is a well-known globular protein that has the tendency to aggregate into macromolecular asmblies [13].X-ray crys-tallography studies have suggested that the native structure of BSA is predominantly a -helical [14].BSA has a wide range of physiological functions inv
olving the binding,transport,and delivery of fatty acids,prophyrins,bilirubin,tryptophan,thyroxine,steroids,metals,pharmaceuticals,and dyes [15].The effectiveness of drugs depends on their
0022-2860/$-e front matter Ó2007Elvier B.V.All rights rerved.doi:10.lstruc.2007.08.020
*
Corresponding author.Tel.:+868172314462;fax:+868172582029.E-mail address: (Y.Zhang).
/locate/molstruc
Available online at
Journal of Molecular Structure 879(2008)
81–87
binding ability.So the studies on the binding of drugs to BSA may provide information of the structural features that determine the therapeutic effectiveness of drugs.How-ever,interaction between OA and bovine rum albumin (BSA)has not been reported,so the study of interaction between OA and BSA is very significant.
In the prent work,we have studied in vitro interaction of OA with BSA by using the quenching of intrinsic fluo-rescence of tryptophan residues.The effect of the energy transfer has been studied according to fluorescence reso-nance energy transfer.The binding constants are obtained at different temperatures in the medium of HCl–Tris (pH 7.4)buffer solution.The binding sites and main sorts of binding force have been suggested.In addition,the confor-mational changes of protein are discusd on the basis of CD and FTIR.
2.Experimental 2.1.Materials
cret是什么意思
Bovine rum albumin (BSA,esntially fatty acid-free)is purchad from Sino-American Biotechnology Company and is ud without further purification and its molecular weight is assumed to be 68,000.All BSA solutions are pre-pared in the pH 7.4buffer solution,and BSA stock solution has
been kept in the dark at 4°C.Oleanolic acid is of ana-lytical grade,and it is purchad from the National Insti-tute for Control of Pharmaceutical and Products,China;NaCl (analytical grade,0.4mol L À1)is ud to maintain the ion strength at 0.1.Buffer solution (pH 7.40)consisted of Tris (0.1mol L À1)and HCl (0.1mol L À1),and the pH is adjusted to 7.40by adding 0.1mol L À1NaOH when the experiment temperature is higher than 298K.The pH mea-surements are made with a PHS-3C digital pH-meter (Jiangsu,Jiangfen Electroanalytical Instrument Co.,Ltd,China)with a combined glass electrode.All solutions are prepared with doubly distilled water.Sample mass are accurately weighed on a electronic analytical balance
ESJ180-4(Shenyang Longteng Electronic Co.,Ltd,China)with a resolution of 0.1mg.2.2.Apparatus and methods
The fluorescence spectra are measured with a F-2500Spectrofluorimeter (Hitachi,Japan)equipped with a 150W Xenon lamp source and 1.0cm quartz cell and a thermostat bath.Equipped with 1.0cm quartz cells,a Shi-madzu UV-2550double-beam Spectrophotometer (Shima-dzu,Japan)is ud for scanning the UV spectrum at room temperature.
Fluorometric titration experiments: 3.0mL solution containing appropriate concentration of BSA is titr
ated by successive additions of a 5.0·10À4mol L À1ethanol stock solution of OA (to give a final concentration of 1.67·10À6mol L À1–2.00·10À5mol L À1).Titrations are done manually by using microsyringe,and the fluorescence intensity is measured (excitation at 287nm and emission at 348nm).All experiments are measured at three tempera-tures (298,305,310K).
Circular dichroism (CD)measurements are made on a Jasco-810automatic recording Spectropolarimeter (Japan),using a 1mm cell at 298K.The spectra are recorded in the range of 200–260nm and the scan rate is 100nm/min with a respon time of 4s.CD determinations of pure BSA and BSA–drug mixtures are carried out using the solutions of drug at a corresponding concentration as the reference.The results are expresd as ellipticity (mdeg),which is obtained in mdeg directly from the instrument.
FTIR measurements are carried out at room tempera-ture on a Brucker Equinox 55FTIR spectrometer (Ger-many).All spectra are taken via the attenuated total reflection (ATR)method with resolution of 4cm À1and 32scans.Spectra processing procedures:spectra of buffer solution are collected at the same condition.Then,subtract the absorbance of solution from the spectra of sample solu-tion to get the FTIR spectra of proteins.The subtraction criterion is that the original spectrum of protein solution between 1800and 1300cm À1is a smooth straight.3.Results and discussion
地心游记3.1.Fluorescence quenching studies
For macromolecules,fluorescence measurements can give some information of binding of small molecules to protein,such as the binding mechanism,binding mode,binding constants,binding sites,and intermolecular dis-tances.A variety of molecular interactions can result in quenching,including excited-state reactions,molecular rearrangements,energy transfer,ground-state complex for-mation,and collisional quenching.
For fluorescence quenching,the decrea in intensity is usually described by Stern–Volmer equation [16]:F 0=F ¼1þK sv ½Q
ð1
stalemateÞ
Fig.1.Molecular structure of oleanolic acid.
82Z.Cheng,Y.Zhang /Journal of Molecular Structure 879(2008)81–87
where F 0and F are the steady-state fluorescence intensities in abnce and prence of quencher,respectively,K sv reprents the Stern–Volmer quenching constant,and [Q ]reprents the concentration of quencher (OA).Hence,Eq.(1)is applied to determine K sv by linear regression of a plot of F 0/F versus [Q ].
The dynamic and static quenching can be distinguished by their different dependence on temperature.The quench-ing rate constants are expected to decrea with increasing temperature for static quenching.In contrast,the reverd effect is obrved for the dynamic quenching [17].
In this experiment,the concentrations of BSA are stabi-lized at 5.0·10À6mol L À1.The effect of OA on BSA fluo-rescence intensity is shown in Fig.2.As can be en from Fig.2,addition of increasing concentrations of OA caus the decrea in the fluorescence intensity,but the maximum emission wavelength is hardly changed.The result indicates that there is no change in the local dielectric envir
onment.The interaction of OA with BSA is further confirmed by FTIR,CD,and UV–vis absorption spectra.
The calculation of K sv from Stern–Volmer plots has demonstrated that varying temperature has a moderate effect on fluorescence quenching by OA.The values of K sv are found to be 4.72·104(R =0.9989), 3.50·104(R =0.9993),and 3.44·104L mol À1s À1(R =0.9995),respectively,at 298,305and 310K.The results show that the K sv decreas with increasing temperature,which indi-cates that the quenching mechanism of OA–BSA binding reaction is initiated by complex formation rather than by dynamic collision.Therefore,the quenching data are ana-lyzed according to the static quenching equation [17].ðF 0ÀF Þ
À1
¼F À10þK À1a F À1
0½Q
À1
ð2Þ
where K a is the binding constant of OA with biomolecule.The dependence of 1/(F 0ÀF )on the reciprocal value of the
quencher [Q ]À1is linear with the slope which equals to the value of (F 0K a )À1(Fig.3).The corresponding results at dif-ferent temperatures are shown in Table 1.It is shown that the binding between BSA and OA is remarkable and the ef-fect of temperature is small.So the quenching efficiency of OA to BSA is not reduced obviously when temperature ris.
3.2.Analysis of binding equilibria
For the static quenching interaction,if it is assumed that there are similar and independent binding sites in the bio-molecule,the binding constant (K )and the numbers sites (n )can be determined according to the method described by Zhao et al.[18],using the following equation:log F 0ÀF F ¼log K þn log ½Q ð3Þ
where K is binding constant of OA with BSA and n is the number of binding sites per albumin molecule,which can be determined by the slope and the intercept of double log-arithm regression cure of log F 0ÀF
F ÀÁversus log[Q ]bad on the Eq.(3).Table 2gives the result of K and n at different temperatures analyzed in this way for BSA.The correlation coefficients are larger than 0.99,indicating that the assump-tions underlying the derivation of Eq.(3)are satisfactory.The values of n at the experimental temperatures are approximately equal to 1,which indicates that there is one class of binding site to OA in
BSA.
Fig.2.Fluorescence spectra of BSA in the prence of various concen-trations of OA,c (BSA)=5.0l M;c (OA)/(l M),1–9:0;1.67;3.33;5.00;6.67;10.00;13.33;16.67;20.00,respectively.The inrts correspond to Stern–Volmer
plot.
Fig.3.The Lineweaver–Burk plot for the binding of OA with BSA.
Table 1
The binding constants between OA and BSA at different temperatures T (K)K a ·104(L mol À1)R SD ·10À4298 6.530.9981 2.83305 3.510.9987 4.48310
2.91
0.9999
1.71
R is the correlation coefficient for K a values.SD is the standard deviation for the K a values.
Z.Cheng,Y.Zhang /Journal of Molecular Structure 879(2008)81–8783
3.3.Binding mode between OA and BSA
The binding studies are carried out at298,305,and 310K.At the temperatures,BSA does not undergo any structural degradation.Generally,small molecules are bound to macromolecules includes binding modes:hydro-gen bond,van der-waals force,electrostatic force,hydro-phobic interaction force,and so on[19].If temperature change is small,the reaction enthalpy change is regarded as a constant.By van’t Hoffand thermodynamic equation:
ln K¼ÀD H0
RT
asfarasþ
D S0
agogoR
ð4Þ
measuredD G0¼D H0ÀT D S0¼ÀRT ln Kð5Þwhere K is the binding constant at temperature T and R is gas constant.D H0,D G0,and D S0are enthalpy change,free energy change,and entropy change,respectively.ln K ver-sus1/T plot(Fig.4)enabled the determination of D H0, D S0,D G0and the values are summarized in Table2. The negative values of D G0indicate that the binding pro-cess is spontaneous.The negative enthalpy(D H0)and en-
tropy(D S0)values of the interaction of OA with BSA indicate that the acting forces are hydrogen bonds and van der-waals forces[20].
3.4.Energy transfer between OA and BSA
The overlap of the UV absorption spectrum of OA with thefluorescence emission spectrum of BSA is shown in Fig.5.According to Forster non-radiative energy transfer theory(FRET)[21],the rate of energy transfer depends on (1)the relative orientation of the donor and acceptor dipoles,(2)the extent of overlap offluorescence emission spectrum of the donor with the absorption spectrum of the acceptor,and(3)the distance between the donor and the acceptor.The energy transfer effect is related not only to the distance between the acceptor and donor,but also to the critical energy transfer distance,R0,and the effi-ciency transfer theory,E,is studied according to FRET. The value of E is calculated by using the following equation
E¼1Àsg是什么意思
F
F0
¼marker是什么意思
R6
昂立英语R6
þr6
:ð6Þ
In Eq.(6),F and F0are thefluorescence intensities of BSA in prence and abnce of OA,r reprents the distance between donor and acceptor and R0is the critical distance at which transfer efficiency equals to50%.The value of R0 is calculated by using the following equation[22],
R6
¼8:8Â10À25k2nÀ4/0Jð7Þ
where k is an orientation factor dependent on the align-ment of the donor and acceptor dipoles,n is the refractive index,/0is the luminescence quantum yield in the abnce of energy transfer,and J is the overlap between the lumi-
Table2
The binding constants K,binding sites n and relative thermodynamic parameters of the system of OA–BSA at different temperatures
T(K)K·103(L molÀ1)n R a D G0(kJ molÀ1)D H0(kJ molÀ1)D S0(J molÀ1KÀ1)R b 29810.08 1.060.9995À22.84À79.27À189.050.9913 305 5.530.990.9993À21.85
310 2.870.890.9986À20.52
a The correlation coefficient for K values.
b The correlation coefficient for the van’t Hoff
plot. Fig.4.van’t Hoffplot,pH7.40,c(BSA)=5.0l
M.
Fig.5.The overlap of the UV absorption of OA with thefluorescence
emission spectrum of BSA:(a)the UV absorbance spectrum of OA,
c(OA)=10.00l M and(b)thefluorescence spectrum of BSA
c(BSA)=5.0l M.
84Z.Cheng,Y.Zhang/Journal of Molecular Structure879(2008)81–87
