Influence of Cd2+, Hg2+ and Pb2+ on (+)-catechin binding to bovine serum albumin studied by fluorescence spectroscopic methods

Spectrochimica Acta Part A 85 (2012) 190–197

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Influence of Cd2+ , Hg2+ and Pb2+ on (+)-catechin binding to bovine serum albumin studied by fluorescence spectroscopic methods Mijun Peng a,b , Shuyun Shi b,c,∗ , Yuping Zhang b a b c

Key Laboratory of Hunan Forest Products and Chemical Industry Engineering, Jishou University, Zhangjiajie 427000, China School of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China Key Laboratory of Resources Chemistry of Nonferrous Metals, Central South University, Changsha 410083, China

a r t i c l e

i n f o

Article history: Received 25 July 2011 Received in revised form 18 September 2011 Accepted 28 September 2011 Keywords: (+)-Catechin Bovine serum albumin Heavy metal ion Fluorescence quenching Interaction

a b s t r a c t The effect of heavy metal ions, Cd2+ , Hg2+ and Pb2+ on (+)-catechin binding to bovine serum albumin (BSA) has been investigated by spectroscopic methods. The results indicated that the presence of heavy metal ions significantly affected the binding modes and binding affinities of (+)-catechin to BSA, and the effects depend on the types of heavy metal ion. One binding mode was found for (+)-catechin with and without Cd2+ , while two binding modes – a weaker one at low concentration and a stronger one at high concentration were found for (+)-catechin in the presence of Hg2+ and Pb2+ . The presence of Cd2+ decreased the binding affinities of (+)-catechin for BSA by 20.5%. The presence of Hg2+ and Pb2+ decreased the binding affinity of (+)-catechin for BSA by 8.9% and 26.7% in lower concentration, respectively, and increased the binding affinity of (+)-catechin for BSA by 5.2% and 9.2% in higher concentration, respectively. The changed binding affinity and binding distance of (+)-catechin for BSA in the presence of Cd2+ , Hg2+ and Pb2+ were mainly because of the conformational change of BSA induced by heavy metal ions. However, the quenching mechanism for (+)-catechin to BSA was based on static quenching combined with non-radiative energy transfer irrespective of the absence or presence of heavy metal ions. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Serum albumin is the major soluble protein in circulatory system, which has many physiological functions, such as maintaining osmotic pressure and pH of blood and as carriers transporting a great number of endogenous and exogenous compounds such as fatty acids, amino acids, drugs and pharmaceuticals [1]. Therefore, it is important to study the interactions of drugs with serum albumin, which determines their pharmacology and pharmacodynamics, and it is also useful to explain the relationship between the structure and function of drugs. Up to now, many manuscripts have reported the interaction between drugs and serum albumin [2–4], and serum albumin has also been implicated in the transport and storage of many metal ions [5,6]. Bovine serum albumin (BSA) has always been selected as the protein model because of its structural homology with human serum albumin (HSA) [7]. Flavonoids are the important phytonutrient components widely distributed in plant foods [8,9]. Numerous investigations of flavonoids in recent years described their beneficial biological

∗ Corresponding author at: School of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China. Tel.: +86 731 88879616; fax: +86 731 88879616. E-mail address: [email protected] (S. Shi). 1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2011.09.059

activities, such as antioxidant activity [10], anti-inflammatory activity [11], antifungal activity [12] and antitumor activity [13]. Therefore, flavonoids have been linked with many health benefits, and foods rich in flavonoids have attracted most interest in recent years. (+)-Catechin (Fig. 1) is among the most widely consumed flavonoids, which exists in many fruits [14], and catechin-type flavonoids present in many common foods [15–17]. Metal ions are widely distributed in nature and can enter human body through air, water, soil, food and many other ways. Unfortunately the metal ions, especially the heavy metals, cannot be degraded in the environment. The low concentrations of heavy metal ions can be enriched and accumulated through the food chain then put highly toxic to living organisms [18]. Moreover, certain human populations are exposed to higher levels of cadmium (Cd2+ ), mercuric mercury (Hg2+ ) and lead (Pb2+ ) today via the ingestion of contaminated food and drinking water than ever before [19,20]. Therefore, many methods have been ascribed to the analysis, monitoring and removal of heavy metal ions from food and drinking water and their risk assessment [21–23]. Recently, the toxic interaction of heavy metal ions with serum albumins has been investigated [24]. After entering the blood, heavy metal ions can bind to anionic groups on plasma proteins through charge interactions and with sulfhydryl groups through coordinate-covalent binding to form complexes. Cd2+ and Hg2+ have high affinity for the sulfhydryl groups of albumin [25,26], while Pb2+ can interact

M. Peng et al. / Spectrochimica Acta Part A 85 (2012) 190–197

191

(4 × 10−3 mol L−1 ) was prepared by dissolving them in Tris–HCl buffer solution, respectively.

OH OH

2.4. Fluorescence spectra

HO

O

OH OH

(+)-Catechin Fig. 1. Molecular structure of (+)-catechin.

with carbonyl and amino groups of albumin [27]. It was well known that the formation of metal ions–BSA complexes could change the binding affinities between drugs and serum albumin. The presence of Cu2+ or Fe3+ affected the binding affinities of ligands with BSA because of the existence of competition binding, conformational change of BSA or metal–ligand chelation [28–31]. However, very little information is available on whether or not the heavy metal ions affected the transportation or disposition of drug in blood [32]. Herein, in view of the biological importance, it was worthwhile to study the effects of heavy metal ions, such as Cd2+ , Hg2+ and Pb2+ on the binding of (+)-catechin to bovine serum albumin (BSA), in order to find out the toxic ions/drug interference. 2. Methods and materials 2.1. Chemicals and reagents (+)-Catechin with the purity over 99.0% was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). BSA was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The other chemicals such as buffer Tris with the purity more than 99.5%, CdCl2 , Hg(NO3 )2 , Pb(NO3 )2 , NaCl, HCl, and ethanol were all of analytical purity and used without further purification, which were all purchased from Sinopharm Chemical Reagent Co. Ltd., China. Water used in all experiments was doubly distilled water. 2.2. Apparatus All fluorescence spectra were recorded on an F-2000 spectrofluorimeter equipped with 1.0 cm quartz cells and a 150 W xenon lamp (Hitachi, Tokyo, Japan). An excitation wavelength of 280 nm was used. The excitation and emission slit width were both set at 2.5 nm. The UV spectra were obtained on a Perkin-Elmer Lambda 17 UV spectrophotometer with the wavelength range of 200–450 nm (Perkin Elmer Corp., Edison, NJ, USA). The weight measurements were performed on an AY-120 electronic analytic weighing scale with a resolution of 0.1 mg (Shimadzu, Japan). The pH value was measured in a pHs-3 digital pH meter (Shanghai, China). 2.3. Preparation of solutions Tris–HCl buffer solution (0.1 mol L−1 Tris, pH 7.4) containing 0.1 mol L−1 NaCl was prepared to keep the pH value and maintain the ionic strength of the solution. The working solution of BSA (1 × 10−4 mol L−1 ) was prepared by dissolving it in Tris–HCl buffer solution and stored in refrigerator at 4 ◦ C prior to use. The (+)-catechin stock solutions (2 × 10−4 mol L−1 ) were prepared by dissolving them in ethanol. The Cd2+ , Hg2+ and Pb2+ stock solution

300 ␮L of BSA solution (or 300 ␮L of BSA solution and 750 ␮L Cd2+ or Hg2+ solution or 120 ␮L Pb2+ ) was added to eleven 5 mL flasks, respectively. After reaction at 25 ◦ C for 1 h, appropriate amounts of 2.0 × 10−4 mol L−1 (+)-catechin were added, and diluted to 5 mL with Tris–HCl buffer. The final concentrations of (+)-catechin were 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 ␮mol L−1 , and the concentration of Cd2+ or Hg2+ was 600.0 ␮mol L−1 , and that of Pb2+ was 96.0 ␮mol L−1 . The resultant mixtures were then incubated at 25 ◦ C for 1.0 h. Then the fluorescence emissions spectra were scanned in the range of 290–450 nm and the fluorescence intensity at 340 nm was measured. All the experiments were repeated in triplicate and found to be reproducible within the experimental error (<1%). 2.5. Determination of binding parameters For the dynamic quenching, the fluorescence quenching data are described by Stern–Volmer equation [33]: F0 = 1 + KSV [Q ] = 1 + kq 0 [Q ] F

(1)

where F0 and F denote the steady state fluorescence intensities of serum albumin with and without quencher, respectively. KSV is the Stern–Volmer quenching constant with the unit of L mol−1 , and [Q] is the concentration of the quencher with the unit of mol L−1 . Stern–Volmer equation was applied to determine KSV by linear regression of a plot of F0 /F against [Q]. kq is the quenching rate constant with the unit of L mol−1 s−1 , while  0 is the average lifetime of the serum albumin without any quencher and is generally equal to 5 ns [31]. The binding constant (K) and binding sites (n) are calculated by the double-logarithm equation for static quenching [34]: log

F − F  0 F

= log K + n log[Q ]

(2)

The efficiency energy (E) was determined by Förster’s energy transfer theory [35]: E =1−

R6 F = 6 0 F0 R0 + r 6

(3)

where F and F0 are the fluorescence intensities of BSA with or without (+)-catechin, r is the distance between acceptor and donor and R0 is the critical distance, which is evaluated as following when the transfer efficiency is 50%: R06 = 8.8 × 10−25 k2 N −4 ˚J

(4)

where k2 is the orientation factor, N is the refractive index of the medium, ˚ is fluorescence quantum yield of the donor. For BSA, k2 = 2/3, N = 1.36 and ˚ = 0.14 [31]. J is overlap integral of the fluorescence emission spectrum of donor and absorption spectrum of the acceptor, which is approximately given by the following equation:

∞ F()ε()4  J = 0 ∞ 0

F()

(5)

where F() is the fluorescence intensity of the donor, while ε() is the molar absorption coefficient of the acceptor. All the above data points were fit to curves by means of OriginPro 7.5 software (OriginLab Corp., Northampton, MA).

192

M. Peng et al. / Spectrochimica Acta Part A 85 (2012) 190–197

800

1.6

a

2+

700

Pb -BSA-(+)-catechin 2+ Hg -BSA-(+)-catechin 2+ Cd -BSA-(+)-catechin (+)-catechin-BSA

1.5

(a) 600 1.4

F0/F

Intensity (F)

BSA (6 uM) + (+)-catechin 500

k

(k)

400

1.2

300

1.1

200 100 0

1.3

1.0 1 300

320

340

360

380

400

420

2

3

4

5 6

440

6

7

8

9

10

-1

10 [Q]/(mol.L )

Wavelength (nm) Fig. 2. The fluorescence quenching spectrum of BSA at various concentrations of (+)-catechin. ex , 280 nm; c(BSA), 6.0 ␮mol L−1 ; c((+)-catechin) (a → k), 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 ␮mol L−1 ; T, 298 K.

Fig. 3. The Stern–Volmer plots for BSA fluorescence quenching by (+)-catechin in the absence and presence of Cd2+ , Hg2+ and Pb2+ at 298 K.

3. Results and discussion

the fluorescence quenching process of BSA at investigated concentrations of (+)-catechin might be mainly governed by a static quenching mechanism rather than a dynamic quenching process.

3.1. Fluorescence quenching of BSA by (+)-catechin

3.2. Fluorescence quenching of BSA by Cd2+ , Hg2+ and Pb2+ ions

BSA has three linearly arranged, structurally distinct, homologus domains (I–III), and each domain is composed of two subdomains (A and B). The specific sites binding with BSA are sites I and II which are located in hydrophobic cavities in the IIA and IIIA subdomains [36]. When the fluorescence emission spectra of BSA are measured with a series of concentrations of quencher by fixing the excitation wavelength at 280 nm, the fluorescence emission peak of BSA at 340 nm gives the information of tryptophan residues [37]. Therefore, fluorescence quenching can be considered as a technique for measuring binding affinities. In the experiment, the sum of the absorbance at 280 nm (excitation wavelength) and 340 nm (fluorescence peak) could cause less than 5% percentage error for each system, therefore, inner filter effect was ignored [6]. The fluorescence of BSA quenched by various concentrations of (+)-catechin is as shown in Fig. 2. It was observed that the fluorescence intensity of BSA dropped regularly with the increasing concentrations of flavonoids, which indicated that the interaction had happened between flavonoids and BSA. About 35.2% of the fluorescence intensities of BSA was quenched by adding 10 ␮mol L−1 (+)-catechin (calculated from Fig. 2). The plots of F0 /F for BSA versus (+)-catechin concentrations exhibited a good linearity (Fig. 3), and the value of kq for (+)-catechin was 1.19 × 1013 L mol−1 s−1 (Table 1), which was far greater than the expected maximum dynamic quenching constant (2.0 × 1010 L mol−1 s−1 ), therefore,

Metal ions could bind with amino, carbonyl, sulfhydryl or hydroxyl groups in BSA to form metal–BSA complex. Moreover, BSA has a negative charge in the experimental conditions [38], which then can bind with metal ions with electrostatic attraction. The effects of Cd2+ , Hg2+ and Pb2+ on the fluorescence of BSA are shown in Fig. 4. The results indicated that the increasing concentrations of Cd2+ hardly changed the fluorescence intensity of BSA, however, the fluorescence intensity of BSA decreased remarkably with the increasing concentration of Hg2+ . Pb2+ quenched more BSA fluorescence than Hg2+ by adding the same concentration; however, the lower concentrations for Hg2+ were selected in the experiments because of the lower solubility for HgCl2 . No obvious spectral shift was observed for heavy metal binding with BSA. For further discussion, the concentrations of Cd2+ and Hg2+ were fixed as 600.0 ␮mol L−1 , while the concentration of Pb2+ was fixed as 96.0 ␮mol L−1 . 3.3. Fluorescence quenching of BSA induced by (+)-catechin in the presence of Cd2+ , Hg2+ and Pb2+ ions Fig. 5 shows the fluorescence spectra of (+)-catechin in the presence of Cd2+ , Hg2+ and Pb2+ . When (+)-catechin was added into BSA solution containing heavy metal ions, further attenuation in the fluorescence of BSA was observed in comparison with metal–BSA system. The em and shape at different concentrations

Table 1 Stern–Volmer quenching constants (KSV ) for the interactions of (+)-catechin with BSA in the presence and absence of Cd2+ , Hg2+ and Pb2+ ions at 298 K.

Free Cd2+ (+)-Catechin–BSA system

Hg2+ Pb2+

a b

R is the correlation coefficient. SD is the standard deviation.

c (␮mol L−1 )

KSV (×104 L mol−1 )

kq (×1012 L mol−1 s−1 )

Ra

SDb

≤10 ≤10 ≤7 ≥7 ≤7 ≥7

5.97 4.98 4.46 5.84 2.57 3.79

11.94 9.96 8.92 11.68 5.14 7.58

0.9950 0.9971 0.9949 0.9901 0.9917 0.9962

0.008 0.012 0.011 0.017 0.008 0.005

M. Peng et al. / Spectrochimica Acta Part A 85 (2012) 190–197

800

A

a

k

700

a b (a) BSA (6 uM) (b)

600

Intensity (F)

800 700

(a)

600

2+

BSA (6 uM) + Cd

2+

500

500

(k)

Intensity (F)

A

193

400 300

BSA(6 uM) + Cd (600 uM) + (+)-catechin

l

(l)

400 300

200 200 100 100 0

300

320

340

360

380

400

420

440 0

Wavelength (nm)

B

300

320

340

360

800

B

800 700

600

440

(a) BSA (6 uM)

2+

BSA (6 uM) + Hg

(b)

k

500

600

400 300

BSA(6 uM) + Hg (600 uM) + (+)-catechin

500

(l)

400

200

300

100

200

0

2+

b

(k)

Intensity (F)

Intensity (F)

420

a

(a)

l

100 300

320

340

360

380

400

420

440

Wavelength (nm)

0 300

800

320

340

360

380

400

420

440

Wavelength (nm)

a

C 700

800

a

(a)

k

b

700

600

(a) BSA (6 uM)

2+

BSA (6 uM) + Pb

(b) 600

500

2+

(k)

Intensity (F)

Intensity (F)

400

a

700

C

380

Wavelength (nm)

400 300

500

BSA(6 uM) + Pb (96 uM) + (+)-catechin

l

(l)

400 300

200

200

100

100 0

300

320

340

360

380

400

420

440

Wavelength (nm) Fig. 4. The fluorescence quenching spectrum of BSA at various concentrations of Cd2+ (A), Hg2+ (B) and Pb2+ (C). ex , 280 nm; c(BSA), 6.0 ␮mol L−1 ; c(Cd2+ ) = c(Hg2+ ) (a → k), 0.0, 60.0, 120.0, 180.0, 240.0, 300.0, 360.0, 420.0, 480.0, 540.0, 600.0 ␮mol L−1 ; c(Pb2+ ) = 0.0, 12.0, 24.0, 36.0, 48.0, 60.0, 72.0, 84.0, 96.0 ␮mol L−1 ; T, 298 K.

0 300

320

340

360

380

400

420

440

Wavelength (nm) Fig. 5. The fluorescence quenching spectrum of BSA at various concentrations of (+)-catechin in the presence of Cd2+ (A), Hg2+ (B) and Pb2+ (C). ex , 280 nm; c(BSA) = 6.0 ␮mol L−1 ; c(Cd2+ ) = c(Hg2+ ) = 600.0 ␮mol L−1 ; c(Pb2+ ) = 96.0 ␮mol L−1 ; c((+)-catechin) (a → k), 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 ␮mol L−1 , T, 298 K.

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M. Peng et al. / Spectrochimica Acta Part A 85 (2012) 190–197

of (+)-catechin in the presence of heavy metal ions were similar to those in the absence of heavy metal ions. When the concentration of (+)-catechin reached 10 ␮mol L−1 , the fluorescence intensities of BSA decreased 33.0%, 32.7% and 22.9% in the presence of Cd2+ , Hg2+ and Pb2+ , respectively. Comparison of the percentages with that obtained in the absence of heavy metal ions showed that the presence of Cd2+ and Hg2+ decreased slightly the quenching effect of BSA fluorescence induced by (+)-catechin, and the presence of Pb2+ decreased considerably the quenching effect induced by (+)catechin. From this point, it can be seen that the quenching process of (+)-catechin for BSA was easily affected by Pb2+ , which may be caused by the highest quenching effect of Pb2+ to BSA. The Stern–Volmer plots for the fluorescence quenching by (+)catechin in the presence of heavy metal ions are shown in Fig. 3. The Stern–Volmer curve for (+)-catechin in the presence of Cd2+ was linear; however, the Stern–Volmer plots for (+)-catechin in the presence of Hg2+ and Pb2+ were nonlinear, and the points could be resolved into two straight lines, which indicated that the interaction between BSA and (+)-catechin in the presence of Hg2+ and Pb2+ followed two types of binding sites. Hg2+ and Pb2+ had higher quenching effect to BSA than Cd2+ , and then the existence of Hg2+ and Pb2+ would change the conformation of BSA more greatly, which could change the binding mode of (+)-catechin to BSA.

C

0.40 0.35

Absorbance

0.30

0.35

c

0.30

0.25

b 0.20 0.15 0.10

a c

0.25

b 0.20 0.15 0.10

d

d

0.05 0.00 250

0.40

a

Absorbance

A

In the linear range of Stern–Volmer curves, all the average kq were far greater than 2.0 × 1010 L mol−1 s−1 (Table 1), which indicated that all the fluorescence quenching were probably originated from the formation of (+)-catechin–BSA complex for static quenching procedure. Therefore, the presence of Cd2+ , Hg2+ and Pb2+ could not change the quenching mechanism of (+)-catechin. UV–vis absorption spectroscopy is a very simple but effective method to explore the structural change and to recognize the complex formation [39]. Fig. 6 shows the UV–vis absorption spectra of BSA and the difference absorption spectra between BSA–(+)catechin and (+)-catechin at the same concentration in the absence or presence of Cd2+ , Hg2+ and Pb2+ , from which we could see that two spectra could not be superposed within experimental error, which reconfirmed that the probable quenching was mainly a static quenching procedure and at least a BSA–(+)-catechin complex with certain new structure formed [40]. From the slopes of the curves for KSV , it could be concluded that the presence of Cd2+ decreased KSV for (+)-catechin by 16.6%, however, the presence of Hg2+ and Pb2+ decreased KSV for (+)catechin by 25.3% and 56.9% in lower concentration, respectively, and decreased KSV for (+)-catechin by 2.2% and 36.5% in higher concentration, respectively. The results indicated that different types of heavy metal ion affected binding interaction between (+)-catechin

0.05

260

270

280

290

300

310

320

330

0.00 250

340

260

270

280

Wavelength (nm)

B

D

0.40 0.35

a b

0.25

Absorbance

Absorbance

0.30

c 0.20 0.15 0.10

300

310

320

330

340

320

330

340

0.40 0.35

a

0.30

c

0.25

b 0.20 0.15 0.10

d

0.05 0.00 250

290

Wavelength (nm)

d 0.05

260

270

280

290

300

310

Wavelength (nm)

320

330

340

0.00 250

260

270

280

290

300

310

Wavelength (nm)

Fig. 6. UV–vis spectra of BSA–(+)-catechin in the absence (A) and presence of Cd2+ (B), Hg2+ (C) and Pb2+ (D). (a) The absorption spectra of BSA–(+)-catechin system without or with corresponding heavy metal ions when the BSA and (+)-catechin were at the same concentration; (b) the absorption spectra of BSA without or with corresponding heavy metal ions; (c) the difference absorption spectra between BSA–(+)-catechin and (+)-catechin at the same concentration; d: the absorption spectra of (+)-catechin only; c(BSA) = c((+)-catechin) = 6.0 ␮mol L−1 ; c(Cd2+ ) = c(Hg2+ ) = 600.0 ␮mol L−1 ; c(Pb2+ ) = 96.0 ␮mol L−1 .

M. Peng et al. / Spectrochimica Acta Part A 85 (2012) 190–197

6.5

-0.2

logK = 0.419 + 4.373n (R = 0.9943)

2+

Pb -BSA-(+)-catechin 2+ Hg -BSA-(+)-catechin 2+ Cd -BSA-(+)-catechin taxifolin-BSA

-0.4 -0.6

6.0

-0.8

log K

log [(F0-F) /F]

195

-1.0

5.5

5.0 -1.2 -1.4

4.5

-1.6 -6.0

-5.8

-5.6

-5.4

-5.2

-5.0

4.0 0.8

0.9

1.0

log[Q]

1.1

1.2

1.3

1.4

n

Fig. 7. Double-logarithm curves of (+)-catechin quenching BSA fluorescence in the absence and presence of Cd2+ , Hg2+ and Pb2+ at 298 K.

Fig. 8. The relationship between log K and n for (+)-catechin to BSA.

and BSA differently, and the quenching process of (+)-catechin for BSA was more easily affected by Pb2+ .

sites (n) with higher correlation coefficient (R = 0.9943) (Fig. 8), which confirmed that mathematical model used in the experiment was suitable to study the interaction between (+)-catechin and BSA. As shown in Table 2, the presence of Cd2+ decreased the binding affinities of (+)-catechin for BSA by 20.5%. The presence of Hg2+ and Pb2+ decreased the binding affinity of (+)-catechin for BSA by 8.9% and 26.7% in lower concentration, respectively, and increased the binding affinity of (+)-catechin for BSA by 5.2% and 9.2% in higher concentration, respectively. The changed binding affinity indicated that the ionic interactions played important roles in (+)catechin–BSA binding in the presence of heavy metal ions. In the experiment, heavy metal ions were first added into the BSA solution for 1 h, and then (+)-catechin was added, therefore, heavy metal ions first combined with BSA to form metal–BSA complex, and then (+)-catechin reacted with metal–BSA complex. The changed binding affinities of (+)-catechin for BSA may be concluded that competitive binding effect between (+)-catechin and heavy metal ions happened, and another reason may be that a conformational change of BSA happened because of binding with different sites, and the third reason probably was a newly formed complex between (+)-catechin and free heavy metal ions, which affect the fluorescence quenching. For (+)-catechin, the likely chelation site for metal ions was the 3 -4 hydroxyl group. The appearance of new peaks in UV–vis absorption spectra was possible to determine whether or not the (+)-catechin–metal complex was formed. The chelating experiments were conducted at pH 7.4. Compared the UV spectra for (+)-catechin in the presence of Cd2+ , Hg2+ and Pb2+ with that in the absence of heavy metal ions (figures not included here), there was no obvious change of absorption maximum for (+)-catechin between 300 and 450 nm in the presence of Cd2+ , Hg2+ and Pb2+ , which indicated that the formation of (+)-catechin–metal complex did not happen.

3.4. Binding affinities of (+)-catechin to BSA in the absence and presence of Cd2+ , Hg2+ and Pb2+ ions Determination of the level of drug binding with serum albumin is critical and will directly correlate with the transport, disposition and in vivo efficacy of the drug. If a drug had low ability to bind with serum albumin, the amount of drug available to diffuse into the target tissue may be significantly reduced, and the efficacy of the drug may then be poor, and vice versa [41]. The binding constants (K) and binding sites (n) can be calculated by the double-logarithm equation. Plots of log(F0 − F)/F versus log[Q] for (+)-catechin–BSA without and with Cd2+ , Hg2+ and Pb2+ are shown in Fig. 7, and Table 2 lists the corresponding calculated results in diverse modes with all the correlation coefficient over 0.99. It is clear that the double-logarithm curves for (+)-catechin in the absence and presence of Cd2+ were linear, but for (+)-catechin in the presence of Hg2+ and Pb2+ were divided into two straight lines. In the presence of Hg2+ and Pb2+ , binding constants and binding sites for (+)-catechin in lower concentration were smaller compared with those in higher concentration. The results indicated that (+)-catechin in lower concentration might facilitate to bind to the binding sites with lower affinity. With the increasing concentration of (+)-catechin, the binding sites with high affinity may be occupied rapidly in view of kinetics, which might facilitate the yield of (+)-catechin–BSA complex, and the second part of the curve my be correlative with these reactions [6]. The values of binding constants of (+)-catechin to BSA in the absence or presence of Cd2+ , Hg2+ and Pb2+ were in the range of 104 –107 mol L−1 , which agreed with the common affinities of drugs for serum albumin [2–4], and the values of log K are proportional to the binding

Table 2 The static binding constants and number of binding sites for the interactions of (+)-catechin with BSA in the presence and absence of Cd2+ , Hg2+ and Pb2+ ions at 298 K.

Free Cd2+ Quercetin–BSA system

Hg2+ Pb2+

a b

R is the correlation coefficient. SD is the standard deviation.

c (␮mol L−1 )

log K

n

Ra

SDb

≤10 ≤10 ≤7 ≥7 ≤7 ≥7

5.76 4.58 5.25 6.06 4.22 6.29

1.19 0.97 1.12 1.28 0.86 1.36

0.9972 0.9990 0.9977 0.9902 0.9933 0.9963

0.027 0.014 0.024 0.019 0.036 0.009

0.35

700

0.35

0.30

600

0.30

700 (a)

500

0.25

400

0.20

300

0.15

C

400

0.20

300

0.15 0.10 (b)

(b)

100 300

320

340

360

380

400

420

440

0.05

100

0.00

0

0.05 300

320

340

Wavelength (nm)

D

0.00

700

0.35

0.30

600

0.30 (a)

500

0.25

400

0.20

300

0.15

Intensity (F)

Intensity (F)

(a)

0.10

200

500

0.25

400

0.20

300

0.15 0.10

200

(b)

(b)

100 320

440

0.35

700

300

420

0.40

0.40

0

400

800

800

600

380

Wavelength (nm)

Absorbance

B

360

Absorbance

0

0.25

(a)

200

0.10

200

500

Absorbance

0.40

0.40

600

Intensity (F)

800

800

Intensity (F)

A

M. Peng et al. / Spectrochimica Acta Part A 85 (2012) 190–197

Absorbance

196

340

360

380

400

420

440

0.05

100

0.00

0

0.05 300

320

340

Wavelength (nm)

360

380

400

420

440

0.00

Wavelength (nm)

Fig. 9. Overlaps of the fluorescence spectra of BSA (a) with the absorption spectra of (+)-catechin (b) with or without heavy metal ions. (A) (+)-catechin–BSA system; (B) (+)-catechin–Cd2+ –BSA system; (C) (+)-catechin–Hg2+ –BSA system; (B) (+)-catechin–Pb2+ –BSA system.

Table 3 J, E, R0 and r values of (+)-catechin with BSA in the absence and presence of Cd2+ , Hg2+ and Pb2+ ions. J (cm3 L mol−1 )

(+)-Catechin–BSA system

Free Cd2+ Hg2+ Pb2+

−14

2.96 × 10 3.02 × 10−14 3.11 × 10−14 3.06 × 10−14

3.5. Effect of Cd2+ , Hg2+ and Pb2+ ions on the binding mode and binding distances for (+)-catechin with BSA

R0 (nm)

r (nm)

25.42 19.23 19.60 13.63

2.79 2.80 2.82 2.81

3.34 3.56 3.57 3.82

d

1.8 1.6

a

1.4

Absorbance

To further ascertain the binding mode of (+)-catechin with BSA in the presence of Cd2+ , Hg2+ and Pb2+ , the binding distances between the donor and acceptor were calculated according to the Förster non-radiation energy transfer theory. As shown in Fig. 9 and Table 3, all the values of r are much smaller than 7 nm and 0.5R0 < r < 1.5R0 , which suggested that the non-radiative energy transfer from BSA to (+)-catechin may occur with high possibility in the presence or absence of Cd2+ , Hg2+ and Pb2+ . So the quenching mechanism for (+)-catechin to BSA was the static quenching combining with non-radiative energy transfer whatever the presence or absence of Cd2+ , Hg2+ and Pb2+ . It is obviously that the values of r for distances between (+)-catechin and BSA in the presence of Cd2+ , Hg2+ and Pb2+ increased. The results were reasonable to assume that Cd2+ , Hg2+ or Pb2+ and (+)-catechin had noncompetitive binding in different albumin sites, which then led to the formation of a ternary nonfluorescent complex, metal–BSA–(+)-catechin. In this situation, the first formation of metal–BSA complex changed the

E (%)

1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 200

225

250

275

300

325

350

Wavelength (nm) Fig. 10. UV spectra of the Hg2+ –BSA system; c(Hg2+ ) (a → d), 0, 400, 500, 600 ␮M.

M. Peng et al. / Spectrochimica Acta Part A 85 (2012) 190–197

conformation of BSA, and then made it easier or more difficult for (+)-catechin to bind with BSA. The change of UV–vis absorption spectroscopy of BSA could explore the structural change. The UV–vis absorption spectroscopy of BSA displayed a strong band with a maximum at about 220 nm and a weak band at about 280 nm, and the peak in 220 nm in the difference spectra of proteins is related to changes of the conformation associated with the helix-coil transformation [30]. As shown in Fig. 10 (only for Hg2+ –BSA system), the absorbance intensity of Hg2+ –BSA system increased with the increasing concentration of Hg2+ and the peak has an obvious red shift from 226 nm to 238 nm, which indicated that the interaction of Hg2+ with BSA may cause the conformational change of BSA. The results were in agreement with that discussed from binding distance r above. 4. Conclusions The effect of toxic heavy metal ions, Cd2+ , Hg2+ and Pb2+ on the binding of (+)-catechin to BSA has been investigated by spectroscopic techniques. The presence of heavy metal ions changed the binding constants and binding modes of (+)-catechin to BSA, and the results indicated that different types of heavy metal ions affected binding process between (+)-catechin and BSA differently. The conformational change of BSA may the main reason for the changed binding affinity and binding distance of (+)-catechin for BSA in the presence of Cd2+ , Hg2+ and Pb2+ . The results provided important insight into the effect of toxicity of heavy metal ions on drug binding with protein which may be correlated to its bioavailability and efficacy. Acknowledgements This work was supported by National Scientific Foundation of China (21005089), freedom explore Program of Central South University (201012200015), Shenghua Yuying project of Central South University, open fund of State Key Laboratory of Powder Metallurgy and aid program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province. References [1] B.X. Huang, H.Y. Kim, C. Dass, J. Am. Soc. Mass Spectrom. 15 (2004) 1237–1247. [2] T.S. Singh, S. Mitra, Spectrochim. Acta A: Mol. Biomol. Spectrom. 78 (2011) 942–948. [3] O. Khani, H.R. Rajabi, M.H. Yousefi, A.A. Khosravi, M. Jannesari, M. Shamsipur, Spectrochim. Acta A: Mol. Biomol. Spectrom. 79 (2011) 361–369. [4] R.G. Machicote, M.E. Pacheco, L. Bruzzone, Spectrochim. Acta A: Mol. Biomol. Spectrom. 77 (2010) 466–472.

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