Binding of fullerol to human serum albumin: Spectroscopic and electrochemical approach

Journal of Photochemistry and Photobiology B: Biology 108 (2012) 34–43

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Binding of fullerol to human serum albumin: Spectroscopic and electrochemical approach Mei-Fang Zhang a, Zi-Qiang Xu a, Yu-Shu Ge a, Feng-Lei Jiang a,⇑, Yi Liu a,b,⇑ a State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China b Department of Chemistry and Life Sciences, Xianning University, Xianning 437005, PR China

a r t i c l e

i n f o

Article history: Received 15 November 2011 Received in revised form 13 December 2011 Accepted 15 December 2011 Available online 23 December 2011 Keywords: Binding Polyhydroxylated fullerene Human serum albumin Fluorescence spectroscopy Electrochemical approach

a b s t r a c t The potential impact of human exposure to carbonaceous nanomaterials in the environment becomes a concerning issue. Here we report on the interaction of fullerol with human serum albumin (HSA) using spectroscopic and electrochemical methods. The water-soluble fullerene derivative (fullerol) was synthesized and characterized by IR, 1H NMR, TG-DSC, XRD, HR-TEM, etc. The spectroscopic methods show that the fluorescence quenching of HSA by fullerol is the result of the formation of an HSA-fullerol complex. Binding parameters such as DG, DH and DS were calculated, and the quenching constant Ka at different temperatures was determined using the modified Stern–Volmer equation. The electrochemical experiments further confirmed the conclusions. In addition, the influences of coexisting heavy metal ions have also been studied in the present system. The circular dichroism spectra (CD), 3D fluorescence spectra and FT-IR spectra results suggest that the secondary structure of HSA was changed by fullerol. Based on the site marker competitive experiments, we can predict the possible binding position of fullerol on the HSA was located at the site of sub domain II A. Furthermore, the distance r between donor (HSA) and acceptor (fullerol) was obtained according to the famous fluorescence resonance energy transfer (FRET) mechanism. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Carbon nanomaterials such as fullerenes have shown remarkable capabilities due to their unique properties. These materials are capable as electron donors or acceptors, and they exhibit photophysical and photochemical behavior, which have imparted to them a number of favorable characteristics, such as magnetic property [1], super-conductivity [2], electrical behavior [3], and biochemical characteristics [4] and so on [5]. In addition, watersoluble fullerenes have gained popularity because of their applications in medicinal and biological systems. These materials are also essential for many emerging biomedical technologies that exploit the unique chemical properties and physical structure of fullerene [6,7]. However, the exposure pathways of most nanomaterials and toxicity mechanisms remained considerably speculative [8]. The correlation between protein-nanoparticle interactions and nanotoxicity is an issue of concern [9].

⇑ Corresponding authors. Address: State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China. Tel.: +86 27 68756667; fax: +86 27 68754067. E-mail addresses: fl[email protected] (F.-L. Jiang), [email protected] (Y. Liu). 1011-1344/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2011.12.006

As the largest amount of soluble and the most important drugcarrying protein in the circulatory system, HSA is involved in the distribution and metabolism of numerous endogenous and exogenous ligands, including fatty acids, amino acids, metal ion and numerous pharmaceuticals [10]. Among these exogenous ligands, nanoparticles that can bind to albumins and then be transported within the circulatory system tend to be easily overlooked. Nanomaterials were shown to enter animals through different routes and transfer between the various organs of the body, although there are still some assumptions that need further research [11]. Consequently, the investigation on the interaction between nanomaterial and HSA is of great importance. Fullerol, a polyhydroxylated fullerene derivative, shows excellent solubility and stability in aqueous solution [12]. There are several ways to synthesize the fullerol, and the simplest method by far, is based on the phase-transfer of C60 in benzene or toluene to an aqueous solution of NaOH, where the polyhydroxylation takes place [13]. However, the final product obtained was shown to be a complex of paramagnetic salt rather than simple polyhydroxylated fullerenes. In the current paper, a modification of a more mature method by Chiang et al. [14] was utilized to achieve high production of the fullerol. Yield was more than 80% with an average number of OH groups above 18. It is no doubt heavy metal ions is one of the worldwide environment problems and can cause various fatal

M.-F. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 108 (2012) 34–43

diseases such as contaminate the oil and aqueous waster stream, even do harm to human’s health [15]. The research of the binding of common heavy metal ions to proteins is of great importance in biological system, and this work is to explore the relationship between them in a superficial level. Although there is considerable interest in the interaction of HSA and fullerenes, the previous literature has litter report related to aqueous solution under physiological condition [12]. In the present work, fluorescence spectroscopy, UV–vis absorption, CD spectroscopy, 3D fluorescence spectra and FT-IR spectroscopy, together with electrochemical approach, were employed to investigate indepth the interaction between HSA and fullerol under physiological condition. This study aims not only to provide valuable information for appropriately understanding the toxicological action of water-soluble fullerene in humans, but also illustrate its binding mechanisms at the molecular level to elucidate the importance of nanomaterial toxicology.

2. Materials and methods 2.1. Materials HSA (purity > 99%) and warfarin were obtained from Sigma–Aldrich (St. Louis, MO, USA), Ibuprofen was obtained from Hubei Biocause Pharmaceutical Co., Ltd (Hubei, China; the purity no less than 99.7%), Crystalline fullerene (C60) powder of 99.9 wt.% purity was purchased from Yongxin Chemical Reagent Company. Fuming sulfuric acid, anhydrous diethyl ether, anhydrous diethyl ether– CH3–CN and all other reagents and solvents used in synthesis and analysis were of analytical reagent grade and purchased from Sinopharm Group Chemical Reagent Company Ltd., Shanghai, PR China. 2.2. Apparatus The LS-55 spectrofluorophotometer from Perkin-Elmer equipped with a thermostat bath was used to measure all of the fluorescence spectra. The absorption spectra of the synthesized fullerol and HSA were recorded by an UNICO 4802 UV–vis double-beam spectrophotometer. The CD spectra were recorded on Cirsular Dichroism Photomultiplier from Applied Photophysics Limited, UK. The electrochemical properties studied were based on a CHI660C electrochemical workstation from Shanghai Chenhua Apparatus Company, PR China. For characterizations, infrared spectra (used in characterization of fullerol) were recorded on an Avatar 360 FT-IR spectrophotometer as KBr pellets. TGA analyses were performed in nitrogen with a temperature scanning rate of 10 K/ min in a thermal analyzer of the NETZSCH STA 449C system. 1H NMR spectra were recorded on mercury 600 MHz NMR spectrometer (Varian, Inc.) using the DMSO-d6 as the solvent. X-ray diffraction (XRD) measurements were performed in the refection mode (Cu Ka radiation, k = 1.5418 Å) on a D8X Advance X-ray diffractometer. The morphology and microstructure analysis of the samples were observed by a high-resolution transmission electron microscope (JEOLJSM-2010) operated at 200 kv. FT-IR spectra were recorded at room temperature on VERTEX 70 spectrometer (Bruker, Germany). 2.3. Preparation of water soluble fullerene fullerol The polyhydroxylated fullerene derivatives were synthesized via hydrolysis of polycyclosulfated precursors. Experimental details for preparation the water soluble fullerol are described in the Supporting Information.

35

2.4. Fluorescence spectral measurements All fluorescence spectra were measured on an LS55 fluorophotometer (Perkin-Elmer Co., USA) equipped with a 1.0 cm quartz cell and a thermostat bath. The widths of the excitation slit and the emission slit were set to 15 nm and 12 nm separately. An excitation wavelength of 280 nm was used throughout to minimize the contribution of the tyrosine residues to the emission. HSA solution was prepared on the basis of its molecular weight of 67,000 and kept in a refrigerator at 4 °C and dissolved in PBS (pH 7.4) at the concentration of 1  105 mol L1. The site-competitive replacement experiments and the effect of coexistent of heavy metal ions experiments were conducted by adding fullerol to the HSA-site marker system and the HSA-heavy metal ions systems separately. 2.5. UV visible absorption spectra and circular dichroism spectra The UV–visible absorption spectra were measured with a 1 cm quartz cell. The concentration of HSA and fullerol were 1.0  105 mol L1. The CD spectra were recorded on Cirsular Dichroism Photomultiplier from Applied Photophysics Limited, UK, using a cylindrical cuvette with 0.1 cm of path-length under the condition of pH (7.4 ± 0.1) at 25 °C. The spectra were recorded from 200 to 260 nm, and the molar ratio of HSA/fullerol was varied as 0:0, 1:1, 1:10, 1:25 and 1:40. 2.6. FT-IR measurements Infrared spectra were collected at room temperature on VERTEX 70 spectrometer (Bruker, Germany) equipped with a zinc selenide (ZnSe) attenuated total reflectance (ATR) accessory, a deuterated triglycine sulfate (DTGS) detector, and a KBr beam splitter. The protein concentration was 0.25 mmol L1, and the molar ratios of drug to protein (cdrug/cHSA) were 1.0. To minimize the effects of water vapor on the IR spectra, the chamber containing the ATR cell was continuously purged with dry air. According to the previous procedures, the infrared spectra of the free protein and drug-protein mixture were collected respectively. Subtraction was performed between the spectrum of HSA in buffer solution and that of the buffer solution to obtain the free protein spectrum with the subtraction criterion that the straight baseline was obtained between 2000 and 1750 cm1 [16]. 2.7. Electrochemical impedance spectroscopy detection 2.7.1. Protein immobilization The electrochemical analyzers were performed with a threeelectrode configuration (CHI 660C). The working electrode was a bare or HSA-modified gold disk electrode (3 mm in diameter); a Pt wire and the Ag/AgCl electrode were utilized as counter and reference electrode, respectively. The HSA was immobilized to the Au surface using the ‘‘dry-adsorption’’ method. Firstly, the working electrode was polished on chamois for 15 min and cleaned in acetone and ultrapure water respectively, then dried in nitrogen airflow. Then a tiny drop (approximately 4–5  106 L) of HSA solution (1  105 mol L1) was dropped to the bare gold surface. At last, the electrode was washed by ultrapure water drop by drop several times and dried in nitrogen airflow after 18 h without hitting, and then the immobilization of HSA tiny layer was fabricated. 2.7.2. Electrochemical measurements The electrolyte used in our experiments is a mixture of K3Fe(CN)6/K4Fe(CN)6 (5 mmol L1) and KCl (10 mmol L1) at pH 7.4. The EIS were measured within the frequency range from 0.1 to 10,000 Hz using the method of titration. Roughly, in order to ensure the success of the modification, firstly the HSA modified

M.-F. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 108 (2012) 34–43

Au electrode was measured in the electrolyte to maintain balance for 15 min. Then different amount of the fullerol (10  103 mol L1) in aqueous solution was added continuously to the system and stirred for 1 min before testing to obtained the EIS spectra in Fig. 5. 3. Results and discussion 3.1. Characterizations of FC The detailed characterizations of fullerol C60(OH)n (n = 19) can be found in supporting information. The 1H NMR (Fig. S2) results and the IR spectral (Fig. S1) show the existence of the hydroxyl groups. The TG-DSC (Fig. S3) results are taken to determine the average number of hydroxyl group. In this work, an exact 19 hydroxyl groups are taken in all experimental content which can be seen from the schematic representation in Fig. 1a. The TEM image (Fig. 1b) of fullerol indicates much less agglomerated particles with average size of 10–30 nm which is in accordance with the result of XRD (Fig. S4) by the Scherrer formula [17]. 3.2. The binding mechanism between fullerol and HSA A variety of molecular interactions can result in quenching. The mechanisms involved include excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, collisional quenching, and so on [18]. The different mechanisms of quenching are usually classified as either dynamic or static quenching, which can be distinguished by their differing dependence on temperature and viscosity, or excited-state lifetime. Since higher temperatures will typically result in larger diffusion coefficients, the bimolecular quenching constants are expected to increase with increasing temperature. By contrast, increased temperature is likely to result in decreased stability of complexes, and thus lower values of the static quenching constants [19]. In this experiment, the HSA solution concentration was stabilized at 1  105 mol L1, and the concentrations of fullerol were varied from 0 to 2.5  105 mol L1 at increments of 0.25  105 mol L1. The fluorescence spectra of HSA obtained in the presence of increasing amounts of fullerol are shown in Fig. 2. The addition of fullerol caused a gradual decrease in the fluorescence intensity of HSA, whereas fullerol alone (curve L) caused slight changes. To study the quenching process by fullerol more closely, fluorescence tests were performed at different temperatures. For fluorescence quenching, the decrease in intensity is usually described by the well-known Stern–Volmer equation [20] (Eq. (1)):

F0 ¼ 1 þ kq s0 ½Q  ¼ 1 þ K SV ½Q  F

ð1Þ

where F0 and F denotes the steady-state fluorescence intensities in the absence or presence of quencher (fullerol), respectively, kq is the biomolecular quenching constant, s0 is the lifetime of the fluorescence in the absence of quencher, KSV is the Stern–Volmer quenching constant and [Q] is the concentration of the quencher. Hence, Eq. (1) was applied to determine KSV by linear regression of a plot of F0/F against [Q]. The calculation of KSV from Stern–Volmer plots (Fig. 3a and Table 1) demonstrated the effect on fluorescence quenching by fullerol at each temperature (298, 304 and 310 K) were investigated; the result indicates that the Stern–Volmer quenching constant KSV decreased with the increasing the temperatures, which means the interactions between fullerol and HSA reaction are initiated by compound formation rather than by dynamic collision. To reconfirm if the probable quenching mechanism of fluorescence of HSA by fullerol is initiated by ground-state complex formation, we used the difference absorption spectroscopy to obtain spectra, the UV–vis absorption spectra of HSA and the difference in the absorption spectra of fullerol-HSA and fullerol at the same concentrations. As can be seen from Fig. 4, curve D (the difference of absorption spectra between [fullerol-HSA] and fullerol) is obviously different from curve B (the absorption spectra of HSA alone), specifically at around 230 nm. These results confirm that the quenching is mainly a static quenching process and is primarily caused by the complex formation between fullerol and HSA [21].

Fluorescence Intensity (a.u.)

36

600 500

A K

400 300 200 100

L(FC only)

0 300

320

340

360

380

400

420

440

Wavelength (nm) Fig. 2. Emission spectra of HSA in the presence of various concentrations of fullerol 298 K. c(HSA) = 1  105 mol L1, c(fullerol)/(105 mol L1), A–K: from 0.0 to 2.5 at increments of 0.25. The curve L shows the emission spectrum of fullerol only under the same condition.

Fig. 1. Schematic representation of (a) fullerol. (b) The TEM of fullerol.

37

M.-F. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 108 (2012) 34–43

EIS experiments were performed on a freshly prepared solution of electrolyte (K3Fe(CN)6/K4Fe(CN)6 (5 mmol L1) and KCl (10 mmol L1)). Comparing the EIS of bare Au electrode (Fig. 5[A]A) and HSA-modified Au electrode (Fig. 5[B]B, we can easily find that the electron transportation become hard due to the successfully immobilization of HSA. Fig. 5[B] shows the results of the corresponding experiment with increasing concentration of FC in the Au electrode that modified by HSA (Fig. 5[B]C–J) under condition of equilibrium interaction. The EIS in Fig. 5[B] reveals a steady decrease in Rct by adding FC to the electrochemical system continuously, which can be seen from the trend of the semicircle in Fig. 5[B]. The total resistance also decreases. This trend and the changes in the UV–vis experiments indicate that the mechanism between the interaction of FC and HSA is a new complex formation process, in contrast to collision. The electronic conductivity of the HSA-modified electrode increased and the new complex may be able to promote electron transfer because of the role of the p–p stacking in binding [22]. For accurate detect the influence of the effect of promotion by adding FC, the affinity constant (KA) was determined by recording equilibrium binding to the probe surface at different target concentrations c0 through the Longmuir adsorption isotherm [23] (Eq. (2)):

(A)

2.0

298K 304K 310K

1.8

F0 /F

1.6 1.4 1.2 1.0

0.0

0.5

1.0

1.5

2.0

2.5

5

10 [Q](moI/L)

(B)

310K 304K 298K

F0/(F0-F)

16

12

Rct ¼ ðRct Þmax  c0 

4

a plot of c0/Rct as a function of c0 yields a straight line from which the affinity constant KA = 1.53  104 L mol1 was deduced as shown in Fig. 6, and the linear correlation reached to 99.36%. KA is a little lower than Ka (Table 2), this difference should be attributed to the temperature while the EIS experiments took place under the temperature of 302 K in the ambience. In all, this value is greatly in agreement with the above result from the fluorescence technique within experimental error. The fluorescence quenching measurements, the changes in UV– vis spectras together with the EIS experiments together confirm that the functionalization between FC and HSA follows static quenching process which forms new complex rather than dynamic collision. Therefore, the quenching data were analyzed according to the modified Stern–Volmer equation [24] (Eq. (3)):

0 0.0

0.5

1.0

1.5

2.0 -5

2.5

10 [Q] 9.9 9.8

3.0

3.5

4.0

4.5

-1

(C) InKa =-2.86+3789.49/T; R= 99.93%

9.7

InKa

KA 1 þ c0  K A

8

ð2Þ

9.6

F0 F0 1 1 1 þ ¼ ¼ DF F 0  F fa K a ½Q fa

9.5 9.4 9.3 3.24

3.28

3.32

3.36

-1

1000/T(K ) Fig. 3. (A) Stern–Volmer plots for the quenching of HSA by fullerol at different temperatures. (B) The modified Stern–Volmer plots for the quenching of HSA by fullerol at different temperatures. (C) Plots of van’t Hoff equation of fullerol and HSA at different temperatures, pH = 7.4, c(HSA) = 1  105 mol L1.

ð3Þ

where DF is the difference in fluorescence intensity between the absence and presence of quencher at concentration [Q], fa is the mole fraction of solvent-accessible fluorophore, and Ka is the effective quenching constant for the accessible fluorophores. The dependence of F0/DF on the reciprocal value of the quencher concentration [Q]1 is linear, with slope equal to the value of (fa Ka)1. The value fa1 is fixed on the ordinate. The constant Ka is the quotient of the ordinate fa1 and the slope (fa Ka)1. Fig. 3b displays the modified Stern–Volmer plots, and the corresponding value of Ka at different temperatures are presented in Table 2. The decreasing trend of Ka with increasing temperatures is accordance with Ksv’s

Table 1 Stern–Volmer quenching constants for the interaction of FC with HSA at various temperatures.

a b

pH

T (K)

104Ksv (L T mol1)

1012kq (L mol1 S1)

R1a

S.D.1b

7.4

298 304 310

4.072 3.905 3.775

0.9901 0.9965 0.9951

0.9901 0.9965 0.9951

0.050 0.029 0.033

R1 is the correlation coefficient. S.D.1 is the standard deviation.

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M.-F. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 108 (2012) 34–43

C

3.0

Y=2650.61-40.60X, R=-0.9936

2400 -1

1.5 1.0 D

0.5

2000

-10

C0/Rct /(10

Absorbance

2.0

MΩ )

A-FC B-HSA C-[FC-HSA] D-[FC-HSA]-FC

2.5

A

1600

1200

B

0.0

800 200

250

300

350

400

0

5

10

Wavelength (nm) Fig. 4. Effect of FC on ultraviolet c(HSA) = c[FC] = 1  105 mol L1 (298 K).

absorption

spectra

of

-Z''/ohm

400

ln K ¼  200 0 750

1000

1250

1500

Z'/ohm

(B)

1400

B

40

DH DS þ RT R

J

1000 800 600 400 200 0 0

1000

45

2000

3000

4000

Z'/ohm Fig. 5. Electrochemical impedance spectroscopy (EIS) of fullerol and HSA system. [A] EIS of bare Au electrode (A), [B] EIS of HSA modified Au electrode (B) and various volume of FC dropping into the HSA (C–K). The volume of B–J (lL): 0, 5, 10, 15, 20, 25, 30, 35, and 40. c(FC) = 10  103 mol L1 in aqueous solution.

dependence on temperature as mentioned above, which coincides with the static quenching mechanism.

In general, the interaction forces between drugs and protein may include electrostatic interactions, multiple hydrogen bonds,

ð5Þ

The corresponding results were presented in Table 2, while the values of DH and DS obtained of the binding site from the slops and ordinates at the origin of fitted lines. The values of DH and DS are found to be 31.507 kJ mol1 and 23.78 J mol1 K1, which indicated that the multiple hydrogen bonds and van der Waals forces played major role in the process of forming the FCHSA complex. The negative sign for DG means that the binding process is spontaneously driven by enthalpy and entropy together. Moreover, previous studies [26] showed that during the conjugation of nanoparticles with protein, other types of forces such as hydrophobic interactions and coordination binding might work besides electrostatic interactions. 3.4. Confirming the binding number and the active sites on HSA 3.4.1. Binding number When small molecules bind independently to asset of equivalent sites on a macromolecule, binding number between them has also been studied through the following double-logarithmic equation [27] (Eq. (6)):

lg 3.3. The determination of the force acting between fullerol and HSA

ð4Þ

where K is the analogous to the effective quenching constants at the corresponding temperatures and R is the gas constant. The temperatures used in our experiment were 298, 304 and 310 K. From Fig. 3c we can see that the linear relationship between ln K and 1/ T is good. The enthalpy change (DH) is obtained from the slope of the van’t Hoff relationship, while the free energy change (DG) is then calculated from the following relationship (Eq. (5)):

DG ¼ DH  T DS ¼ RT ln K

1200

-Z''/ohm

35

van der Waals interactions, hydrophobic and steric contacts within the antibody-binding site, etc. [25]. To elucidate the interaction between FC and HSA, the temperature-dependent thermodynamic parameters were calculated from the van’t Hoff plots. It is supposed that the enthalpy change (DH) does not vary significantly in the temperature rang studied and can be considered as a constant, then both the enthalpy change (DH) and entropy change (DS) can be evaluated from the van’t Hoff equation (Eq. (4)):

A

500

30

Fig. 6. Plot of Langmuir absorption isotherm model for fullerol and HSA system.

600

250

25

C0 /(10 M)

HSA.

800

0

20

-6

(A)

1000

15

  F0  F ¼ lg K b þ n lg½Q  F

ð6Þ

where F0 and F stands to the fluorescence intensity in the absence and presence of quencher at various concentration of [Q] respectively, Kb refers to the binding constant, n is the binding number.

39

M.-F. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 108 (2012) 34–43 Table 2 Binding constants and relative thermodynamic parameters of HSA-FC interaction at pH = 7.4.

a b c d

pH

T (K)

104 Ka (Lmol1)

R2a

S.D.2b

DH (kJmol1)

DS (Jmol1K1)

DG (kJmol1)

R2c

R2d

7.4

298 304 310

1.9137 1.4652 1.1700

0.9998 0.9988 0.9986

0.091 0.100 0.294

31.506

23.78

24.427 24.244 24.143

0.9993

0.013

R2 is S.D.2 R2 is R2 is

the correlation coefficient for modified Stern–Volmer plots. is the standard deviation for modified Stern–Volmer plots. the correlation coefficient for van’t Hoff plots. the standard deviation plots for van’t Hoff.

According to the equation, the binding number can be obtained from the slop of the plot of log [(F0  F)/F] vs. log [Q]. Table 3 demonstrates the value of n is close to 1, which suggests the water-soluble fullerene and HSA have a strong binding during their interaction process. 3.4.2. Binding site To identify the FC binding site on HSA, site marker competitive experiments are carried out, using drugs (warfarin and ibuprofen) as site marker fluorescence probes for monitoring site I and site II of HSA respectively. Since FC and HSA has one binding number, to clarify the specific binding location, we used drugs (warfarin and ibuprofen) to bind specifically bind to a known site or region on HSA. Then information about the specific binding site can be gained by monitoring the changes in the fluorescence of FC–bound HSA that brought about by site I (warfarin) and site II (ibuprofen) markers (Fig. 9) [28]. In the site marker competitive experiment, FC was gradually added to the solution of HSA and site markers held in equimolar concentrations (1.0  105 mol L1). As shown in Fig. 7, with the addition of site marker (warfarin and ibuprofen) into HSA, the fluorescence intensity was lower than that of without site markers. In order to facilitate the comparison of the influence of warfarin and ibuprofen in the binding of FC to HSA, the binding constants in the presence of site markers were analyzed according to the modified Stern–Volmer equation (Eq. (2)). The corresponding results are shown in Table 4, which indicates that the ibuprofen has little influence on the binding of FC to HSA, while the binding constant was surprisingly variable in the presence of warfarin. Fig. 7 shows the comparison of the fluorescence spectra of FC-HSA system with the warfarin and ibuprofen. It can be observed that, the fluorescence property of the FC- HSA system was almost the same as in the absence of ibuprofen. By contrast, Fig. 7[A] the maximum emission wavelength of HSA had an obvious red shift, and the fluorescence intensity was significantly higher than that without warfarin. These results indicated that the binding of FC to HSA was obviously affected by adding warfarin, and the polar of the region surrounding the trypyophan site (Try-214) [29,30] was also increased. As discussed above, the results and plots demonstrated that the decrease in probe fluorescence may result from competitive displacement of the probe, and FC bind with high affinity to site I (subdomain IIA) of HSA.

3.5. Energy transfer between fullerol and HSA Taking into account an overlap between the emission spectrum of HSA and the absorption spectrum of fullerol (Fig. S7), an excitation energy transfer mechanism might be assumed. The theory of FRET (Fluorescence Resonant Energy Transfer) [31] is vividly called ‘‘the spectral scale’’, and is widely used in biological systems. For the intrinsic fluorescence of protein is generated by the tryptophan residues, the efficiency of the energy transfer in biochemistry can be used to evaluate the distance r between the ligand and the tryptophan residues in the protein. According to Förster and Sinanoglu’s [32] energy transfer theory, the efficiency of energy transfer between the donor and acceptor, E, could be calculated using equation (Eq. (7)):

E¼1

F R6 ¼ 6 0 F 0 R0 þ r 6

ð7Þ

where F and F0 are the fluorescence intensities of HSA in the presence and absence of fullerol, r is the distance between donor and acceptor, and R0 is the critical distance, at which the efficiency of transfer is 50%. R0 can be calculated by using the following equation (Eq. (8)):

R60 ¼ 8:79  1025 K 2 n4 UJ

ð8Þ

where K2 is the orientation factor involved the geometry of the donor–acceptor dipole, n is the refractive index of medium, U stands for the donor’s fluorescence quantum yield, and J expresses the degree of spectra overlap between the donor’s emission and the acceptor’s absorption. J is usually calculated by the following equation (Eq. (9)):

R1 J¼

0

FðkÞeðkÞk4 dk R1 FðkÞdk 0

ð9Þ

where F (k) is the fluorescence intensity of the donor at wavelength range k, e(k) is the molar absorption coefficient of the acceptor at the certain wavelength k. It has been reported that in the biochemistry system, K2 = 2/3, n = 1.36, U = 0.15, J can be evaluated by integrating the overlap spectra in Fig. S7 according to Eq. (7) while J = 6.366  1015 cm3 L mol1. Based on these data, we found R0 = 3.5 nm and r = 0.8R0. Thus, the distance between FC and Try residue in HSA is about 2.8 nm. The donor–acceptor distance

Table 3 Binding number of FC to HSA.

a b

pH

T (K)

n

R4a

S.D.4b

7.4

298 304 310

1.120 1.227 1.151

0.9986 0.9998 0.9996

0.0203 0.0079 0.0112

R4 is the correlation coefficient for double-logarithmic plots. S.D.4 is the standard deviation for double-logarithmic plots.

M.-F. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 108 (2012) 34–43

600

(A)

HSA only

500

A

α -Helix(%)

0

A

MRE (deg.cm2 .dmol-1)

Fluorescence Intensity (a.u.)

40

K

400 300 200 100

warfarin only

A B C D E

-5000

-10000

E -15000

A -20000

0 300

320

340

360

380

400

420

440

200

210

Fluorescence Intensity (a.u.)

wavelength (nm) 600

57.00 56.70 55.50 53.90 45.80

(B)

HSA only A

220

230

240

250

260

Wavelength (nm) Fig. 8. The CD spectra of FC system obtained at room temperature and pH 7.4; c(HSA) = 1.0  105 mol/L; c[fullerol]/(1.0  105 mol/L). A–F: 0, 1, 10, 25 and 40 respectively.

A

500

K 400 300 200 100

Ibuprofen only 0 300

320

340

360

380

400

420

440

wavelength (nm) Fig. 7. Effect of site marker to the FC system (T = 298 K, kex = 295 nm), c(ibuprofen) = c(warfarin) = c(HSA) = 1.0  105 mol L1; c((b-CD)2/C60)/(1.0  105), A–K: 0; 0.25; 0.50; 0.75; 1.00; 1.25; 1.50; 1.75; 2.00; 2.25 and 2.50. The curve in the bottom shows the emission spectrum of warfarin [A] and ibuprofen [B] only in this condition.

conditions of the experiments [34]. Excessive copper uptake can cause serious health problems, such as damage to heart, kidney, liver, pancreas and brain, as well as intestinal distress, and anemia [35]. According to the Ref. [36], HSA has one tryptophan residue Trp-214, located in sub-domain IIA. In our experiment, we have predicted the possible binding position of fullerol on the HSA was located at the site of sub domain II A. So we think it is the proper ionic radius that as a major factor may decide which ion can enter into the cavity of the HSA and has a stronger binding. Among the Mn, Co and Ba ions, the Cu ion has a median ionic radius. Of course, the electronic structure, the preferred coordination geometry, and these ions between itself and the combination of strength and proteins may also have an impact. Therefore, given that copper is one of the heavy metal ions that cause protein denaturation, the interactions between FC, HSA, and copper discussed in the present paper should awaken public attention. 3.7. The conformation change of HSA induced by fullerol

(0.5R0 < R0 < 1.5R0) and r is in the range of 2–8 nm indicate that the energy transfer from HSA to FC occurs with a high probability [33].

3.6. The effect of coexistent of heavy metal ions The binding of common heavy metal ions to proteins is of great importance in biological science, which is related to human health and the environmental development. The higher binding constants obtained in the presence of heavy metal ions might result in prolonging the storage time of FC in the blood plasma and enhancing the risk of FC sequestration by organs because serum albumin is the major transport protein in the circulation system. The influences of different heavy metal ions on the FC-HSA system are diverse (Table 5). Among these ions, copper ion has the highest binding constant, which may be ascribed to the synergy between pH value and the toxicity of the copper ion under the physiological

3.7.1. CD spectra Fig. 8 shows the CD spectra of HSA and the FC-HSA complex obtained at pH 7.4. The CD spectra of HSA displays two minima in the ultraviolet region, one at 208 nm and the other at 222 nm, which is characteristic of the a-helical structure of a protein. The CD results were expressed in terms of mean residue ellipticity (MRE) in deg cm2 d mol1 according to the following equation (Eq. (10)):

Table 5 The binding constants of FC system at 298 K in the presence of common heavy metal ions.

Table 4 Parameters of the site competitive replacement experiments.

a b

Site Marker

Ka (104 L mol1)

R5a

S.D.5b

Blank Warfarin Ibuprofen

1.914 1.148 1.671

0.9998 0.9994 0.9967

0.091 0.009 0.231

R4 is the correlation coefficient for modified Stern–Volmer plots. S.D.5 is the standard deviation for modified Stern–Volmer plots.

a

Constant ions

104Ka (L mol1)

Ra

S.D.b

K/K0c

Control Pd2+ Mn2+ Fe3+ Cu2+ Ni2+ Co2+ Zn2+ Hg2+ Cd2+ Ba2+

1.914 7.574 0.327 13.344 20.030 2.515 0.308 2.492 6.307 2.421 0.783

0.9998 0.9973 0.9969 0.9949 0.9971 0.9957 0.9998 0.9981 0.9919 0.9975 0.9977

0.091 1.120 1.001 0.739 1.156 1.776 0.229 0.7052 0.9847 1.3233 0.9480

1.00 3.957 0.171 6.972 10.465 1.314 0.161 1.302 3.295 1.265 0.409

R is the correlation coefficient. S.D. is the standard deviation. c K0 is the binding constant of FC system in the absence of common heavy metal ions. b

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M.-F. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 108 (2012) 34–43

Fig. 9. Three-dimensional flourescence spectra of HSA (A) and FC–HSA complex (B), c (HSA) = c (FC) = 10 mmol L1 (298 K).

observed CDðm degÞ cp nl  10

ð10Þ

where cp is the molar concentration of the protein, n is the number of amino acid residues (585 for HSA) and l is the path length (0.1 cm). The contents of a-helical structure of HSA were calculated from the MRE value at 208 nm using the following equation (Eq. (11)):

a-helix ð%Þ ¼

MRE208  4000 33; 000  4000

characteristics of tryptophan and tyrosine residues, and peak 2 is related to changes the conformation of the peptide backbone with the kex = 200–330 nm, kem = 200–500 nm respectively. Obviously, peak 2 is changed greater than peak 1, so the peptide backbone, tryptophan and tyrosine residues and the conformations all have been altered. Therefore it concludes that there was specific interaction occurring between FC and HSA, FC has complexed with HSA to change its conformation [40].

ð11Þ

where MRE208 is the MRE value observed at 208 nm, 4000 is the MRE of b-form and random coil conformation cross at 208 nm and 33,000 is the MRE value of a pure a-helix at 208 nm. The contents of a-helix and b-strand are calculated by using SELCON3. The results are listed in Table 6. After the titration of FC, the helicity of HSA decreased significantly and the b-strands, turn, and unordered structure increased slightly [37–39]. As known, the secondary structure contents are related closely to the biological activity of protein, thus a decrease in a-helix from 57.0% to 45.8% (Table 6) meant the loss of the biological activity of HSA upon interaction with a higher concentration of FC, which also suggests a structural change. 3.7.2. Three-dimensional fluorescence spectra For further insight into the conformation change of HSA by addition of the water-soluble nanomaterial of fullerol, the threedimensional fluorescence spectra offers another valuable method to monitor the changes in the secondary structure of protein and their dynamics. Fig. S6 (Supplementary information) show the contour spectra of HSA and HSA-FC. From Fig. 9A and B, we can see the two peak regions (peak 1 at 218.27 nm/310.06 nm and peak 2 at 192.52 nm/324.46 nm) were observed. Peak 1 shows the spectral Table 6 Fractions of different secondary structures determined by SELCON3. Molar ratio [FC]:[HSA]

H(r) (%)

H(d) (%)

S(r) (%)

S(d)(%)

Trn

Unrd

0:1 1:1 10:1 25:1 40:1

37.3 36.9 36.1 34.1 27.7

19.7 19.8 19.4 19.8 18.1

2.6 2.8 3.9 4.4 5.0

3.1 3.4 3.8 4.9 5.4

15.4 16.4 16.8 17.6 19.5

21.6 22.4 23.6 24.9 26.8

H(r): regular a-helix; H(d): distorted a-helix; S(r): regular b-strand; S(d): distorted b-strand; Trn: turns; Unrd: unordered structure.

3.7.3. FT-IR spectroscopy Additional evidence regarding the HSA-FC system complications comes from infrared spectroscopy results since infrared spectra of proteins exhibit a number of so-called amide bands that represent different vibrations of the peptide moiety. Of all the amide modes of the peptide group, the single most widely used one in studies of protein secondary structure is amide I [10,41]. In the IR region, the amide I peak position occurred in the region 1600–1700 cm1 (mainly C@O stretch) and the amide II band occurs in the region 1500–1550 cm1 (CAN Sstretch coupled with NAH bending mode) [38,39,41]. Fig. 10 shows the FT-IR spectrum of free HSA (up) and the different spectra of FC-HSA system (down). As can be seen from the infrared spectrum, the peak position of amide I moved from 1656.5 to 1658.5 cm1, and amide II

1656.5

0.016 1546.5 1658.5

0.012

Absorbance

MRE ¼

0.008

1548.6

HSA

0.004 HSA-FC

0.000 1800

1750

1700

1650

1600

1550

1500

Wavelength (nm) Fig. 10. The FT-IR spectra of HSA in the absence and presence of fullerol.

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M.-F. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 108 (2012) 34–43

moved from 1546.5 to 1548.6 cm1 when HSA was interacted with the FC. These results again indicated that the conformation of HSA has been change by the FC during the interaction, which was also in agreement nicely with the results of CD and 3D experiments. 4. Conclusions In this work, we systematically studied the interaction between one water-soluble fullerene fullerol and HSA by fluorescence, CD, FT-IR, UV–vis and electrochemical methods. The studies presented here demonstrated that fullerol’s binding to HSA was a result of the formation of fullerol-HSA complex. The results show that fullerol is a strong quencher of the fluorescence of HSA and binds to the protein with high affinity. The binding parameters were calculated using the modified Stern–Volmer equation. The possible binding position of fullerol on HSA at the subdomain II A site was deduced. Furthermore, the decrease of the a-helix amount, at least on the structure of HSA, may ascribe to the distinct property of the water-soluble fullerol nanoparticles. In addition, a p–p stacking interaction might play an important role in binding, since p–p stacking may be a general mode for the interaction of fullerene and their derivatives with proteins [22]. Our studies about the influence of heavy metal ions on FC system also indicate that understanding the interactions between proteins and nanoparticles are crucial for toxicological studies of C60 nanoparticles. 5. Abbreviations HSA FC NMR CD 3D fluorescence spectra

human serum albumin fullerol nuclear magnetic resonance circular dichroism three-dimensional fluorescence spectroscopy

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