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Spectrochimica Acta Part A 81 (2011) 645– 652

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

j ourna l ho me page: www.elsev ier .com/ locate /saa

nvestigation of the binding of Salvianolic acid B to human serum albumin andhe effect of metal ions on the binding

ingting Chenb, Hui Caob, Shajun Zhuc, Yapeng Lua, Yanfang Shangb, Miao Wangb,anfeng Tangb, Li Zhua,∗

Institute of Nautical Medicine, Nantong University, Nantong 226019, PR ChinaSchool of Chemistry and Chemical Engineering, Nantong University, Nantong 226019, PR ChinaDepartment of General Surgery, Affiliated Hospital of Nantong University, Nantong 226001, PR China

r t i c l e i n f o

rticle history:eceived 4 May 2011eceived in revised form 19 June 2011ccepted 26 June 2011

eywords:alvianolic acid Buman serum albumin

nteractionluorescenceircular dichroism

a b s t r a c t

The studies on the interaction between HSA and drugs have been an interesting research field in lifescience, chemistry and clinical medicine. There are also many metal ions present in blood plasma, thusthe research about the effect of metal ions on the interaction between drugs and plasma proteins iscrucial. In this study, the interaction of Salvianolic acid B (Sal B) with human serum albumin (HSA) wasinvestigated by the steady-state, synchronous fluorescence and circular dichroism (CD) spectroscopies.The results showed that Sal B had a strong ability to quench the intrinsic fluorescence of HSA through astatic quenching mechanism. Binding parameters calculated showed that Sal B was bound to HSA withthe binding affinities of 105 L mol−1. The thermodynamic parameters studies revealed that the bindingwas characterized by positive enthalpy and positive entropy changes, and hydrophobic interactions werethe predominant intermolecular forces to stabilize the complex. The specific binding distance r (2.93 nm)

between donor (HSA) and acceptor (Sal B) was obtained according to Förster non-radiative resonanceenergy transfer theory. The synchronous fluorescence experiment revealed that Sal B cannot lead to themicroenvironmental changes around the Tyr and Trp residues of HSA, and the binding site of Sal B on

HSA is located in hydrophobic cavity of subdomain IIA. The CD spectroscopy indicated the sec-ondary structure of HSA is not changed in the presence of Sal B. Furthermore, The effect of metal ions(e.g. Zn2+, Cu2+, Co2+, Ni2+, Fe3+) on the binding constant of Sal B–HSA complex was also discussed.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

The distribution, metabolism and free concentration of vari-us drugs are strongly affected by drug–protein interactions in thelood stream [1–3]. This type of interaction can also influence therug stability and toxicity during the chemotherapeutic process [1].erum albumins are the abundant and soluble proteins in the circu-atory system. It could play a dominant role in the drug dispositionnd efficacy [4]. Many drugs and bioactive small molecules wouldind reversibly to albumin and other serum components that thenunction as carriers [5]. Serum albumin often increases the appar-nt solubility of hydrophobic drugs in plasma and modulates theirelivery to cell in vivo and in vitro [6]. Consequently, it is important

o study the interaction between drug and serum albumin.

Human serum albumin is the most abundant protein in bloodlasma, which constitutes up to 60% of the total protein corre-

∗ Corresponding author. Tel.: +86 513 85051797; fax: +86 513 85051796.E-mail address: lzhu1965@yahoo.com.cn (L. Zhu).

386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.saa.2011.06.068

sponding to a concentration of 42 g L−1 [7] and provides about 80%of the osmotic pressure of blood [8]. It is a globular protein con-sisting of a single peptide chain of 585 amino acids [9,10], withthree �-helical domains I–III, each containing two subdomains Aand B. The protein is stabilized by 17 disulfide bridges. The crys-tal structure analyses indicate that the principal regions of ligandbinding sites in albumin are located in hydrophobic cavities in sub-domains IIA and IIIA. These binding sites are known as Sudlow Iand Sudlow II, respectively [11,12]. HSA plays an important role inthe transport and disposition of many endogenous and exogenoussubstances such as metabolites, drugs, and other biologically activecompounds present in blood [7]. It is also widely accepted that thedegree of affinity between HSA and the drug can govern the distri-bution of the drugs into tissues, affect the elimination of the drugand consequently affect the therapeutic or toxic effects of the drug[13]. Therefore, the detailed investigation of drug–HSA interaction

would possess the significance for the thorough understanding ofthe pharmacodynamics and pharmacokinetics behavior of the drugand for the design of the analogues with effective pharmacologi-cal properties. However, the trace metal ions, which are stored in

6 ca Acta

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hbScaSapoitti

itiflsstdeFFi

2

2

A(LT(0iHSTtocnd

46 T. Chen et al. / Spectrochimi

lood plasma, may affect the binding reaction of drug and HSA. Sot is also necessary to investigate the interaction between drug andSA both in the presence and absence of metal ions.

Salvia miltiorrhiza (Danshen) is a popular traditional Chineseerb widely used in treating coronary heart disease [14], cere-rovascular disease [15], hepatitis [16] and hepatocirrhosis [17].alvianolic acid B (Sal B, Fig. 1), one of the major water-solubleomponents extracted from S. miltiorrhiza, is the most abundantnd bioactive member of the Salvianolic acids isolated to date from. miltiorrhiza [18]. It was reported that Sal B could reduce thetherosclerosis [19], improve the regional cerebral blood flow [20],rohibit the platelet aggregation [21] and ameliorate the hemorhe-logy parameters [22]. However, the detailed investigation of thenteraction of HSA with Sal B has not been conducted. Because ofhe wide application of Sal B in biomedical science, it is worthwhileo study the interaction between HSA and Sal B. It will give the deepnsight of its clinical pharmacological application.

In order to better understand the mechanism of the drug bind-ng to HSA and get a complete view on the binding process, inhe present study, the interaction between HSA and Sal B at var-ous temperatures was examined by means of the steady-stateuorescence, synchronous fluorescence and circular dichroismpectroscopies. The characteristics of the binding, i.e. binding con-tant, number of binding sites, nature of binding forces, as well ashe effects of the complexation on the protein conformation wereetermined. In addition, the thermodynamic and Förster param-ters associated with the binding process were also calculated.urthermore, The effect of metal ions (e.g. Zn2+, Cu2+, Co2+, Ni2+,e3+) on the binding constant of Sal B–HSA complex was also stud-ed.

. Materials and methods

.1. Materials

Human serum albumin (HSA, purity 96–99%, catalogue No.1653) was purchased from Sigma–Aldrich Chemical Company

St. Louis, MO, USA). Sal B (≥98.0%) was obtained from Zelang Co.td. (Nanjing, China) and was used without further purification.ris(hydromethyl)aminomethane (Tris) was purchased from AcrosGeel, Belgium). The Tris–HCl buffer (0.05 M, pH 7.40) containing.10 M NaCl was selected to keep the pH value and maintain the

onic strength of the solution. The 1.0 × 10−5 M stock solution ofSA was prepared in pH 7.40 Tris–HCl buffer. The stock solution ofal B (1.5 × 10−3 M) was prepared by dissolving Sal B with pH 7.40ris–HCl buffer. For CD spectra, the sodium phosphate buffer solu-ion (20 mM, pH 7.40) was selected according to the requirement

f the apparatus. All other reagents and solvents were of analyti-al grade and used without further purification unless otherwiseoted. All aqueous solutions were prepared using newly double-istilled water.

OHHO

O

O

O

O

OH

OH

COOHO COOH

HO

OH

OH

Fig. 1. Molecular structure of Salvianolic acid B.

Part A 81 (2011) 645– 652

2.2. Fluorescence spectra

Fluorescence spectra were recorded on a RF-5301PC fluorometer(Shimadzu, Japan) equipped with 1.0 cm quartz cell. The emissionspectra were recorded between 290 and 550 nm with an exci-tation wavelength of 280 nm. Excitation and emission slit widthwere 5 nm and 3 nm, respectively. A series of assay samples wereincubated in the dark for 1 h at 298 and 310 K, respectively, andthen the fluorescence intensities were recorded. Synchronous flu-orescence spectra of HSA in the absence and presence of differentamount of Sal B were also recorded by scanning the excitation andemission monochromator simultaneously. The wavelength interval(��) was fixed individually at 15 and 60 nm, at which the spectrumonly showed the spectroscopic behavior of tyrosine and tryptophanresidues of HSA, respectively.

2.3. UV absorption spectra

The ultraviolet visible (UV–vis) absorption spectra wererecorded on a UV-2450 spectrophotometer (Shimadzu, Japan)equipped with 0.5 cm quartz cells. The pH measurements werecarried out on a PHS-3C Exact Digital pH meter (Cole-Parmer Instru-ment Co., IL, USA).

2.4. CD spectra

CD spectra of the samples in the range of 190–250 nm wererecorded on a JASCO J-810 automatic recording spectropolarime-ter (Tokyo, Japan) controlled by the Jasco software with a 0.1 cmquartz cell at room temperature, and the speed of scanning was50 nm/min. The concentration of HSA was 3 �M, and the buffersolution was selected as the blank and was automatically sub-tracted from each spectrum during scanning. Each sample wasscanned for three times to average for a CD spectrum.

3. Results and discussion

3.1. Fluorescence quenching of HSA by Sal B

The fluorescence of HSA originates from the tryptophan (Trp),tyrosine (Tyr), and phenylalanine (Phe) residues [7]. Actually, theintrinsic fluorescence of HSA is almost contributed by the Trpresidue alone, because the Phe has a very low quantum yield andthe fluorescence of Tyr is almost totally quenched when it is ion-ized or near by an amino group, a carboxyl group, or a Trp [23].The characteristic of the intrinsic fluorescence of HSA is very sensi-tive to its microenvironment. It would be quenched obviously evenif there is a slight change of the local surroundings of HSA, suchas biomolecular binding, protein conformation and denaturation.Hence, fluorescence spectra were recorded to assess the interactionof Sal B with HSA.

In the experiment, the concentrations of HSA solution were sta-bilized at 3.0 × 10−6 mol L−1, and the concentrations of Sal B variedfrom 0 to 1.8 × 10−5 mol L−1at increments of 1.5 × 10−6 mol L−1.Fig. 2 shows the fluorescence emission spectra of HSA with thedifferent concentrations of Sal B. Obviously, HSA had a strongfluorescence emission band at 339 nm by fixing the excitationwavelength at 280 nm, which is mainly due to its single Trp residue(Trp-214) [24]. As seen in Fig. 2, the addition of Sal B to HSA causeda gradual decrease in the fluorescence intensity of HSA withoutchanging the maximum emission wavelength and shape of thepeaks, which is interpreted that Sal B could interact with HSA and

quench its intrinsic fluorescence. No spectral shifting was observedfor the emission spectra upon Sal B–HSA complexation, indicatingthat Trp residue is not exposed to any change in polarity [25]. Sub-sequent addition of Sal B provoked an increase in the emission at

T. Chen et al. / Spectrochimica Acta Part A 81 (2011) 645– 652 647

5505004504003503000

100

200

300

400

500Fl

uore

scen

ce In

tens

ity

Wavelength / nm

a

k

a

k

FH1

∼obtc

3

iciflqodfiwlt

q(

wtaSottr

(svqs[

H

1.61.41.21.00.80.60.40.20.0-0.20.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6a

b

F 0/F

[Q] (10-5 M)

298 K310 K

765432101

2

3

4

5

6

7

8

9

10

11

1 / [Q] (105 M-1)

F 0 /

(F0-

F)

298 K 310 K

Fig. 3. (A) Stern–Volmer curves and (B) modified Stern–Volmer curves for the

ig. 2. Effect of Sal B on fluorescence spectra of HSA (T = 298 K, pH 7.4). (a) 3.0 �MSA; (b–k) 3.0 �M HSA in the presence of 1.5, 3, 4.5, 6.0, 7.5, 9, 10.5, 12.0, 13.5,5 �M Sal B.

478 nm due to Sal B bound to the HSA. Moreover, the occurrencef isoactinic point at 428 nm might also indicate the existence ofound and free Sal B in equilibrium [26]. In other words, an isoac-inic point was considered as a direct evidence for drug–proteinomplex formation [27].

.2. Quenching mechanism

Quenching can be classified as either dynamic or static quench-ng by different mechanisms. Dynamic quenching results fromollision between fluorophore and quencher, and static quench-ng is due to the formation of ground-state complex betweenuorophore and quencher [28]. In general, dynamic and staticuenching can be distinguished by their different dependencen temperature and viscosity. Dynamic quenching depends uponiffusion. Since higher temperatures result in larger diffusion coef-cients, the bimolecular quenching constant is expected to increaseith increasing temperature. In contrast, increased temperature is

ikely to result in decreased stability of complexes and thus leadso lower values of the static quenching constants.

In order to confirm the quenching mechanism, the fluorescenceuenching data are analyzed by the well known Stern–Volmer Eq.1) [28] and modified Stern–Volmer Eq. (2) [29].

F0

F= 1 + Kq�0[Q ] = 1 + KSV[Q ] (1)

F0

�F= F0

F0 − F= 1

fa+ 1

faKa[Q ](2)

here F0 and F are the fluorescence intensities before and afterhe addition of the quencher, respectively. Kq, KSV, �0, fa, Ka,nd [Q] are the quenching rate constant of the biomolecule, thetern–Volmer dynamic quenching constant, the average lifetimef the biomolecule without quencher (�0 = 10−8 s [30]), the frac-ion of accessible fluorescence, the effective quenching constant forhe accessible fluorophores, and the concentration of the quencher,espectively.

Within certain concentration, the curve of F0/F versus [Q]Stern–Volmer curve) would be linear if the quenching type is singletatic or dynamic quenching [31]; similarly, the curve of F0/(F0 − F)ersus 1/[Q] (modified Stern–Volmer curve) would linear for staticuenching [32]. If the quenching type is combined quenching (both

tatic and dynamic), the Stern–Volmer plot is an upward curvature33].

Fig. 3(A) displays the Stern–Volmer plots of the quenching ofSA fluorescence by Sal B. The plot shows that within the investi-

quenching of HSA fluorescence by Sal B at different temperatures (298 K and 310 K).CHSA = 3.0 �M; pH 7.4.

gated concentrations, the results agree with the Stern–Volmer Eq.(1). Quenching type should be single static or dynamic quenching.Fig. 3(B) shows the modified Stern–Volmer curves. From Fig. 3(B),it was known that under certain Sal B concentration, the curves ofF0/(F0 − F) versus 1/[Q] were linear. All these results showed thatthere were obviously characters of static quenching.

As a rule, the KSV values decrease with an increase in tem-perature for static quenching, and the reverse effect would beobserved for dynamic quenching [34]; the maximum scatter col-lision quenching constant, Kq,max of various quenchers with thebiopolymer was 2.0 × 1010 L mol−1 s−1 [32]. If the Kq > Kq,max, thefluorescence quenching of biopolymer surely do not come fromdynamic quenching. In this paper, the KSV, and Kq at differenttemperatures were listed in Table 1. It indicated that the KSV val-ues decreased with an increase in temperature and the Kq wasapproximately 1012 L mol−1 s−1. Obviously, this indicated that thequenching was not initiated from dynamic collision but from theformation of a compound. In addition, the corresponding results ofKa values were also listed in Table 1. The decreasing trend of Ka

with increasing temperature was in accordance with KSV’s depen-dence on temperature as mentioned above, which also coincideswith static quenching mechanism.

3.3. The binding constants and the number of binding sites

For the static quenching process, when small molecules bindindependently to a set of equivalent sites on a biomacromolecule,

648 T. Chen et al. / Spectrochimica Acta

-4.8-5.0-5.2-5.4-5.6-5.8-6.0

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

log10 [Q]

log 1

0 [(F

0-F)

/F]

298 K 310 K

Ffl

tf

l

wpncvts

dptB(msucimsB

3

aTaecpt

TS

ig. 4. Plots of log10(F0 − F)/F against log10[Q] for Sal B quenching effect on HSAuorescence at different temperatures.

he equilibrium between free and bound molecules is given by theollowing equation [35]:

og10F0 − F

F= log10 Kb + n log10[Q ] (3)

here F0 and F are the fluorescence intensities in the absence andresence of the quencher, respectively, Kb is the binding constant,

is the number of binding sites per albumin molecule and [Q] is theoncentration of quencher. Fig. 4 showed the plots of log10(F0 − F)/Fersus log10[Q] for the Sal B–HSA system at different temperatures,he calculated binding constants (Kb) and the number of bindingites (n) are presented in Table 2.

The value of Kb is significant to understand the distribution of therug in plasma since the weak binding can lead to a short lifetime oroor distribution, while strong binding can decrease the concentra-ions of free drug in plasma. The binding constants (Kb) between Sal

and HSA were 1.84 × 105 (298 K, R = 0.9998) and 2.00 × 105 L M−1

310 K, R = 0.9988), and the number of binding sites (n) was deter-ined as 1.069 ± 0.011. These results illustrated that there is a

trong binding force between Sal B and HSA, and almost one molec-lar of Sal B binds to one molecular of HSA with high affinity. Theorrelation coefficients are larger than 0.998, indicating that thenteraction between Sal B and HSA agrees well with the site-binding

odel underlying Eq. (3). The value of Kb illustrated that there is atrong binding force between Sal B and HSA, which implies that Sal

can be tightly stored and carried by HSA in the body.

.4. Thermodynamic parameters and nature of the binding forces

Generally, there are essentially four types of non-covalent inter-ction that could play a key role in ligand binding to proteins.hese are hydrogen bonds, van der Waals forces, hydrophobicnd electrostatic interactions. Thermodynamic parameters, free

nergy (�G), standard enthalpy (�H) and standard entropy (�S)an provide an insight into the binding mode [36]. Among thesearameters, �G reflects the possibly of reaction, �H and �S arehe main evidence to determine acting forces. When temperature

able 1tern–Volmer quenching constant (KSV) and modified Stern–Volmer association constant

T (K) Eq. (1)

10−4 KSV (L mol−1) 10−12 Kq (L mol−1 s−1) Ra

298 9.79 9.79 0.9994

310 8.62 8.62 0.9970

a R is the correlation coefficient.b SD is the standard deviation of the fit.

Part A 81 (2011) 645– 652

varies in a small range, the �H can be considered as a constant.Then through the binding constant Kb, thermodynamic parametersare evaluated using the following equations:

�G = −RT ln Kb (4)

lnKb2

Kb1=

[1T1

− 1T2

]�H

R(5)

�G = �H − T �S (6)

where Kb2 and Kb1 are the binding constants at different tempera-ture and R is gas constant. The �H, �G and �S for the interaction ofSal B with HSA at different temperatures are shown in Table 2. Thenegative sign for �G means that the interaction process is sponta-neous. Ross and Subramanian [36] have characterized the sign andmagnitude of the thermodynamic parameter associated with var-ious individual kinds of interaction that may take place in proteinassociation processes. For typical hydrophobic interactions, both�H and �S are positive, while these are negative for van der Waalsforces and hydrogen-bond formation in low dielectric media. More-over, the specific electrostatic interaction between ionic species inan aqueous solution is characterized by positive �S value and nega-tive �H value (very small, almost zero). In the present case, �H and�S for the binding reaction between Sal B and HHb are found to be5.60 kJ mol−1 and 119.56 J mol−1 K−1. The positive �H and �S val-ues indicated that hydrophobic interactions play a major role in theSal B–HSA binding reaction and make contributions to the stabilityof the complex. From the structure of Sal B (Fig. 1), the aromatichydrocarbon can easily integrate into the hydrophobic pocket ofHSA.

3.5. Energy transfer from HSA to Sal B

Fluorescence resonance energy transfer (FRET) has been used asa “spectroscopic ruler” for measuring molecular distances in bio-logical and macromolecular systems [37]. According to the Förster,the efficiency of FRET between donor and acceptor depends mainlyon the following factors: (i) the donor can produce fluorescencelight; (ii) fluorescence emission spectrum of the donor and UV–visabsorbance spectrum of the acceptor have more overlap; and (iii)the distance between the donor and the acceptor approach and islower than 8 nm [38]. Here the donor and acceptor were HSA andSal B, respectively. Using FRET, the distance r between Sal B andHSA could be calculated by the following equation [28].

E = 1 − F

F0= R6

0

(R60 + r6)

(7)

where E denotes the efficiency of transfer between the donor andthe acceptor, r is the average distances between donor and acceptor,and R0 is the critical distance when the efficiency of transfer is 50%.

R60 = 8.79 × 10−25K2N−4˚J (8)

In Eq. (7), K2 is the orientation related to the geometry of the donorand acceptor of dipoles and K2 = 2/3 for random orientation as influid solution; N is the average refracted index of medium in thewavelength range where spectral overlap is significant; ̊ is the

(Ka) of the system of Sal B–HSA at different temperatures.

Eq. (2)

SDb 10−4 Ka (L mol−1) Ra SDb

0.0181 7.36 0.9999 0.03590.0351 5.09 0.9983 0.1693

T. Chen et al. / Spectrochimica Acta Part A 81 (2011) 645– 652 649

Table 2Binding constant Kb and relative thermodynamic parameters of the system of Sal B–HSA.

T (K) Kb (L mol−1) n Ra SDb �H (kJ mol−1) �S (J mol−1 K−1) �G (kJ mol−1)

298 1.84 × 105 1.058 0.9998 0.0068 5.60 119.56 −30.035 5

fltal

J

wic

tEaowwna

3

cavSt

3

mstvflt(

FsU

Trp residue of HSA which decreased about 49.04% in fluorescenceintensity, which indicated that Sal B reached subdomain IIA, wherethe only one Trp residue (Trp-214) in HSA is located [9]. There is a

0

50

100

150

200a

Fluo

resc

ence

Inte

nsity

a

k

310 2.00 × 10 1.079 0.9988 0.017

a R is the correlation coefficient.b SD is the standard deviation of the fit.

uorescence quantum yield of the donor; J is the effect of the spec-ral overlap between the emission spectrum of the donor and thebsorption spectrum of the acceptor (Fig. 5), which could be calcu-ated by the equation:

=∫ ∞

0F(�)ε(�)�4d�∫ ∞0

F(�)d�(9)

here F(�) is the corrected fluorescence intensity of the donorn the wavelength range, from � to � + ��; ε(�) is the extinctionoefficient of the acceptor at �.

In the present case, N = 1.336, ̊ = 0.118 for HSA [39], accordingo Eqs. (7)–(9), we could calculated that J = 7.83 × 10−15 cm3 L mol−1

= 0.216; R0 = 2.35 nm; and the binding distance r = 2.93 nm. Theverage distances between a donor fluorophore and acceptor flu-rophore are on the 2–8 nm scale [40], and 0.5R0 < r < 1.5R0 [41],hich indicate that the energy transfer from HSA to Sal B occursith high probability. In accordance with prediction by Förster’son-radiative energy transfer theory, these results indicated again

static quenching mechanism between Sal B and HSA.

.6. Effect of Sal B on the HSA conformation

We had ascertained that it is the binding of Sal B to HSA thataused the fluorescence quenching of HSA, but it is still a puzzlebout whether the binding affects the conformation and microen-ironment of HSA or not. In order to further verify the binding ofal B to HSA and investigate HSA structure after binding interaction,he methods of synchronous fluorescence and CD were utilized.

.6.1. Synchronous fluorescence studiesSynchronous fluorescence is a kind of simple and effective

eans to measure the fluorescence quenching and the possiblehift of the maximum emission wavelength, which is related tohe alteration of the polarity around the chromophore microen-

ironment. As is well known, the experiment on synchronousuorescence of HSA will provide the characteristic information forhe Tyr residues and Trp residues when the scanning interval ���� = �em − �ex) is fixed at 15 and 60 nm, respectively [42]. When

4404204003803603403203000

100

200

300

400

500

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

b

Fluo

resc

ence

Inte

nsiti

ty

Wavelength / nm

a

Abs

orba

nce

ig. 5. The overlap of the UV–vis absorption of Sal B with the fluorescence emissionpectrum of HSA: (a) the fluorescence spectrum of HSA, CHSA = 3.0 �M and (b) theV–vis absorbance spectrum of Sal B, CSal B = 15.0 �M (T = 298 K, pH = 7.4).

−31.46

�� is set at 15 or 60 nm, the shift of the maximum emission wave-length reveals the alteration of polarity microenvironment aroundTyr or Trp residues [43]. By investigating the synchronous fluores-cence spectra of Tyr residues and Trp residues, we could explorethe conformational changes of HSA. The synchronous fluorescencespectra of interaction between Sal B and HSA are presented in Fig. 6.It was shown that the fluorescence intensity of HSA decreased reg-ularly along with the addition of Sal B, which further demonstratedthe occurrence of fluorescence quenching in the binding process.Moreover, there is no significant shift of the maximum emissionwavelength with �� = 15 nm (Fig. 6(A)) or 60 nm (Fig. 6(B)), whichimplies that interaction of Sal B with HSA does not affect the con-formation of the region around the Tyr or Trp residues. From thesynchronous fluorescence data, we can also get the informationabout the location of binding site. It has been shown in Fig. 7 thataddition of the Sal B resulted in strong fluorescence quenching of

330320310300290280

b

Wavelength / nm

3803603403203000

100

200

300

400

Fluo

resc

ence

Inte

nsity

Wavelength / nm

a

k

Fig. 6. Synchronous fluorescence spectra of interaction between HSA and Sal B (A)at �� = 15 nm and (B) at �� = 60 nm. Concentration of HSA was 3.0 �M while con-centrations of Sal B were 0, 1.5, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5, 12.0, 13.5, 15.0 �M froma to k (T = 298 K, pH = 7.4).

650 T. Chen et al. / Spectrochimica Acta Part A 81 (2011) 645– 652

0.0000150.0000100.0000050.000000

0.5

0.6

0.7

0.8

0.9

1.0 Δλ =15 nmΔλ =60 nm

F/F 0

[Q] / M

F�

lbsSoebcIb2flaht

3

ctopfn2[

Fc(

1.61.41.21.00.80.60.40.20.0-0.20.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

[Q] (10-5 M)

F 0/F

HSA HSA-Zn HSA-Cu HSA-Co HSA-Ni HSA-Fe

ig. 7. The quenching of HSA synchronous fluorescence by Sal B. C(HSA) = 3 �M. (�)� = 15 nm and (�) �� = 60 nm.

arge hydrophobic cavity in subdomain IIA which many drugs canind to [44]. It was also observed from Fig. 7 that the fluorescencetrength of Tyr residues decreased about 49.82% in the presence ofal B. Thus there are almost similar decreasing percentages of flu-rescence intensity for Trp and Tyr residues. These results can bexplained that Trp and Tyr residues of HSA may have equal accessi-ility to Sal B [45,46], and the strong fluorescence quenching in thease of Tyr residue is due mainly to one nearby Tyr in subdomainIA. The location of Tyr-263 residue is in subdomain IIA, close to theinding site and not completely buried inside the protein [47]. Tyr-63 may be one of the residues responsible for the synchronousuorescence spectrum of HSA when �� is fixed at 15 nm [48]. Thebove results indicated that Sal B can bind HSA primarily in theydrophobic cavity of subdomain IIA with higher binding affinityo form a stable Sal B–HSA complex,

.6.2. CD spectra studiesCD is a sensitive technique to monitor the conformational

hanges of protein upon an interaction with a ligand. To ascertainhe possible influence of Sal B binding on the secondary structuref HSA, CD measurement was also performed in the absence andresence of Sal B. Consistent with the literature, the CD spectrum

or HSA (Fig. 8) monitored in the range 250–190 nm exhibited twoegative double humped peaks in the ultraviolet region at 209 and22 nm, which is characteristic of an �-helix structure of protein49]. The negative peaks at 209 and 222 nm are both contributed to

250240230220210200190

-40

-20

0

20

40

60

80

Δε

/ M-1

cm-1

Wavelength / nm

HSA Sal B:HSA=1 Sal B:HSA=3 Sal B:HSA=6

ig. 8. Far UV-CD spectra of HSA in the absence and presence of increasing con-entration of Sal B in the range of 190–250 nm. CHSA = 3.0 �M, CSal B/CHHb = 0, 1, 3, 6T = 298 K, pH = 7.4).

Fig. 9. Stern–Volmer plots for the quenching of HSA fluorescence by Sal B in thepresence of different metal ions (Zn2+, Cu2+, Co2+, Ni2+, Fe3+) at 298 K.

n → �* transfer for the peptide bond of �-helix [50]. However, Fig. 8also reveals that, for the Sal B–HSA system, the appearance of the CDspectra is exactly similar to that of HSA alone, which indicates thatthe structure of HSA after addition of Sal B was also predominantly�-helix. The superimposed CD spectra of the HSA in the absenceand presence of the Sal B reveal that, at least in the experimentalconcentrations range, there is no perceptible secondary structuralchange of the HSA upon binding with the Sal B, thus Sal B hardlyaffects the secondary structure of HSA and can maintain protein sta-bilization. The similar observations were also reported by Banerjeeet al. [51] and Chakrabarty et al. [52].

3.7. Effect of metal ions on the affinity of Sal B for HSA

In plasma, there are some metal ions, which can affect the reac-tions of the drugs and serum albumins. These trace metal ions areessential in the human body and exhibit structural and functionalroles in many biomolecules. Some references report that Cu2+, Ni2+,Zn2+, Co2+ and other metal ions can form complexes with serumalbumin [53–58]. The presence of metal ions in plasma may affectthe interaction of drugs with HSA. The effects of common metal ions(e.g. Zn2+, Cu2+, Co2+, Ni2+, Fe3+) on binding constants of Sal B–HSAcomplex were investigated at 298 K. Sal B also made the intrinsicfluorescence of HSA quenched in the presence of Zn2+, Cu2+, Co2+,Ni2+ and Fe3+, respectively. The Stern–Volmer plots of the quench-ing of HSA fluorescence by Sal B in presence of different metal ionswere shown in Fig. 9, the values of KSV and Kq for each metal ionwere calculated and summarized in Table 3. In the presence of thesemetal ions, the values of Kq for Sal B are far higher than the maxi-mum scatter collision quenching constant (2.0 × 1010 L mol−1 s−1),which suggests that quenching is initiated by a complex formation,

corresponding to a static quenching mechanism [59]. In this case,the interactions can be interpreted by static binding constant (Kb).Fig. 10 showed the plots of log10(F0 − F)/F versus log10[Q] for theSal B–HSA system in presence of metal ions. The values of bind-

Table 3Stern–Volmer quenching constants and quenching rate constants of Sal B–HSA sys-tem at 298 K in the presence of different metal ions.

Zn2+ Cu2+ Co2+ Ni2+ Fe3+

KSV (×104 L mol−1) 7.74 10.08 9.84 9.17 9.57Kq (×1012 L mol−1 s−1) 7.74 10.08 9.84 9.17 9.57Ra 0.9991 0.9986 0.9983 0.9974 0.9984SDb 0.0173 0.0276 0.0299 0.0345 0.0283

a R is the correlation coefficient.b SD is the standard deviation of the fit.

T. Chen et al. / Spectrochimica Acta

-4.8-5.0-5.2-5.4-5.6-5.8-6.0

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

log10 [Q]

log 10

[(F0

-F)/F

]HSA HSA-Zn HSA-Cu HSA-Co HSA-Ni HSA-Fe

Fig. 10. Plots of log10(F0 − F)/F against log10[Q] for Sal B quenching effect on HSAfluorescence intensity in the presence of different metal ions (Zn2+, Cu2+, Co2+, Ni2+,Fe3+) at 298 K.

Table 4The binding constants of Sal B–HSA system at 298 K in the presence of differentmetal ions.

Zn2+ Cu2+ Co2+ Ni2+ Fe3+

K ′b (L mol−1) 9.22 × 104 1.04 × 105 1.23 × 105 1.30 × 105 5.47 × 104

n 1.018 1.006 1.023 1.036 0.954Ra 0.9992 0.9992 0.9989 0.9981 0.9964SDb 0.0141 0.0133 0.0160 0.0219 0.0274K ′ /K 0.50 0.57 0.67 0.71 0.30

iidceTcTdbaTthhcacctsboice

4

whc

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b b

a R is the correlation coefficient.b SD is the standard deviation of the fit.

ng constant and the number of binding sites acquired were listedn Table 4. As shown in Table 4, Zn2+, Cu2+, Co2+, Ni2+ and Fe3+

ecreased the binding constants of Sal B–HSA system by 29–70%ompared to that in the absence of these metal ions, which can bexplained by the transition of HSA molecular conformation mainly.he formation of metal ion-HSA complex of regular geometricalonfiguration would resist HSA to effectively enwrap Sal B [60,61].herefore, It could be concluded that participation of metal ions inrug–HSA binding may affect drug–HSA interaction. The degree ofinding to albumin may have consequences for the rate of clear-nce of metabolites and for their delivery to cells and tissues [62].he conventional view is that the cellular uptake is proportionalo the unbound concentration of drugs. According to the free drugypothesis [63], the Sal B distribution within the body is generallyeld to be driven by the free concentration of unbound Sal B inirculating plasma. The reversible binding to blood proteins, suchs serum albumin, �-acid glycoprotein and lipoproteins, may haveonsequences for the delivery of the Sal B and their metabolites toells and tissues. If a molecule is highly bound to plasma proteins,he amount of drug available to diffuse into the target tissue may beignificantly reduced and the efficacy of the drug may consequentlye poor. Here it was found that metal ions decreased the affinitiesf Sal B for HSA. The metal ions in blood will affect the transport-ng ability of serum albumin for Sal B, which may improve the freeoncentrations of unbound Sal B and enhance the pharmacologyffects of Sal B [26]. These effects are significant in vivo.

. Conclusions

In the present work, the interaction of Salvianolic acid B (Sal B)ith human serum albumin (HSA) under physiological conditionsas been investigated by the steady-state, synchronous fluores-ence and circular dichroism (CD) spectroscopies. The results

[

[[

Part A 81 (2011) 645– 652 651

showed that Sal B had a strong ability to quench the intrinsic fluo-rescence of HSA by forming the Sal B–HSA complex through a staticquenching mechanism. The binding constants (Ka) were 1.84 × 105

and 2.00 × 105 L mol−1 at 298 and 310 K, respectively. The thermo-dynamic standard enthalpy (�H) and standard entropy (�S) for thereaction were calculated to be 5.60 kJ mol−1 and 119.56 J mol−1 K−1,respectively, which illustrated that the hydrophobic interactionplayed a major role in binding of Sal B to HSA. The distancebetween Sal B and the tryptophan residue of HSA was esti-mated at 2.93 nm using Förster’s theory of nonradiative resonanceenergy transfer. The binding site of Sal B on HSA was locatedin hydrophobic cavity of subdomain IIA by the analysis dataof synchronous fluorescence spectra. From synchronous fluores-cence and CD spectra, Sal B hardly affected the conformationof HSA. The presence of metal ions (e.g. Zn2+, Cu2+, Co2+, Ni2+,Fe3+) decreased the binding constant of the Sal B–HSA complexand increased the free concentration of unbound Sal B result-ing in a shortening storage time of the Sal B in blood plasmatogether with an enhancement of the maximum effectiveness ofthe Sal B, and these effects are significant in vivo. The bindingstudy of drugs to physiologically important protein HSA is greatlyimportant in pharmacy, pharmacology and biochemistry, whichmay provide some references for the rational use of drugs in theclinic.

Acknowledgements

We are grateful for the financial support from the National Natu-ral Science Foundation of China (Grant Nos. 30971197, 20906052),the Natural Science Foundation of Jiangsu Province (Grant Nos.BG2007607, BK2010281), and the Science and Technology Bureauof Nantong (Grant No. K2009041).

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