Research Article Received: 9 August 2014,
Revised: 29 October 2014,
Accepted: 14 January 2015,
Published online in Wiley Online Library: 19 February 2015
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2463
Direct interactions in the recognition between the environmental estrogen bisphenol AF and human serum albumin Lijun Yang, Junna Lv, Xin Wang, Jing Zhang, Qi Li, Tingting Zhang, Zhenzhen Zhang and Lei Zhang* Bisphenol AF (BPAF) was used as a model compound to investigate the binding mechanism between the endocrine disrupting compound and human serum albumin (HSA) using multispectroscopic techniques and molecular modeling method at the protein level. The results indicated that BPAF was indeed bound to HSA and located in the hydrophobic pocket of HSA on subdomain IIA through hydrogen bond and van der Waals interactions. The fluorescence quenching data showed that the binding of BPAF and HSA quenched the intrinsic fluorescence of HSA, and the static quenching constants were acquired. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: Bisphenol AF; Human serum albumin; Interaction; Multi-spectroscopic techniques
INTRODUCTION
J. Mol. Recognit. 2015; 28: 459–466
* Correspondence to: Lei Zhang, College of Chemistry, Liaoning University, Shenyang 110036, China. E-mail: [email protected] L. Yang, J. Lv, X. Wang, J. Zhang, Q. Li, T. Zhang, Z. Zhang, L. Zhang College of Chemistry, Liaoning University, Shenyang 110036, China
Copyright © 2015 John Wiley & Sons, Ltd.
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It is well known that endocrine disruptors attract the interests of an increasing number of scientists because of their possible negative effects on human health. Some endocrine disruptors have been reported to be environmental pollutants that can give rise to abnormal sexual development and abnormal feminizing on animals (Ohko et al., 2001). Among these endocrine disruptors, bisphenol A (BPA) has been given special attention (Rochester, 2013; Pan et al., 2014). Bisphenol AF [1,1,1,3,3,3-hexafluoro-2,2-bis(4-hydroxyphenyl)propane] (BPAF), the fluorinated homologue of BPA, has CF3 moiety, which may be much more electronegative and reactive than the CH3 of BPA. Therefore, BPAF may pose high potentiality as an endocrine disruptor for humans and wildlife via binding with hormone receptors (Kitamura et al., 2005). BPAF has broad applications in the areas such as electronic devices, optical fibers, and food processing equipment (Akahori et al., 2008; LaFleur and Schug, 2011). It has become one of the high-yielding chemicals in the world because of its growing demand. Because of its wide usage in the environment and its estrogenic activity in specific responses in vitro and in vivo, it is reasonable to assume that BPAF may be harmful to human health. Binding of xenobiotics to plasma proteins has toxicological importance, because it alters their free, active concentrations, and as a consequence, the degree and time of action in the body also affects the duration and intensity of their effects (Kragh-Hansen, 1981; Cserháti and Forgcás, 1995). BPAF’s effects, however, on serum albumin remain unclear. The affinity between BPAF and serum albumin is an important factor to understand the toxicological properties of BPAF as it strongly influences BPAF distribution and determines the free fraction that is available for subsequent interactions with targeted receptors.
Serum albumin, the most abundant protein in plasma, functions in the maintenance of colloid osmotic blood pressure and in the binding and transportation of various ligands such as fatty acids, hormones, and drugs (Zolese et al., 2000). The distribution, free concentration, and metabolism of these ligands strongly depend on their binding constants with serum albumin (Wu et al., 2007). Human serum albumin (HSA) is an ideal protein to use in studies of endocrine disruptor–protein binding (Westermark et al., 1987; Engel et al., 2008). Therefore, investigating the interaction of BPAF and HSA is significant for understanding its transport and distribution in the body and for clarifying its action mechanisms and pharmacodynamics. As of yet, however, no work has been reported for the mechanism of this interaction and the detailed physicochemical characterizations of BPAF binding to HSA. In this paper, the interaction between BPAF and HSA was systematically studied by multispectroscopic techniques. We estimated the association constants, number of binding sites, thermodynamic parameters, and binding force for the interaction of BPAF with HSA. The specific binding site of BPAF on HSA was investigated in detail. The effect of BPAF on the microenvironment and conformation of HSA was also studied. This study provides basic data for clarifying the binding mechanisms of BPAF with HSA and is helpful for understanding its effect on protein function during its transportation in the blood and its toxicity in vivo.
L. YANG ET AL.
EXPERIMENTAL Reagents and apparatus HSA (CAS#70024-90-7) (purity > 99%) was purchased from Heowns Biochemical Technology Co., Ltd. (Tianjin, China). The analytical standard BPAF (CAS#1478-61-1) (chemical reference substance, 97.0% purity) was purchased from Heowns Biochemical Technology Co., Ltd. (Tianjin, China). Ketoprofen and ibuprofen were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). All other chemicals were of analytical reagent grade. Ultrapure water used throughout experiments was purified using a Sartorius Arium 611 system (Sartorius AG, Göttingen, Germany). All fluorescence measurements were carried out through a Cary Eclipse fluorescence spectrophotometer (Varian Inc., Palo Alto, CA, USA) equipped with a thermostat bath and a 1-cm quartz cell. The absorption spectra were recorded on the ultraviolet–visible (UV–Vis) Cary 5000 spectrophotometer (Varian Inc., Palo Alto, CA, USA) equipped with two 1-cm quartz cells. Circular dichroism (CD) measurements were performed on a J-810 spectro-polarimeter (JASCO Corp., Tokyo, Japan) using a 1-cm quartz cell. Fourier-transform infrared spectroscopy (FT-IR) measurements were carried out at room temperature on a Prestige-21 FT-IR spectrometer (SHIMADZU Inc., Kyoto, Japan).
The CD spectra of HSA (4.0 × 107 mol l1) in the presence of BPAF were recorded in the range of 200–250 nm at 298 K under constant nitrogen flush. The molar ratio of BPAF to HSA was varied as 0:1, 5:1, 10:1, and 20:1, and each CD spectrum was the average of three successive scans. Three-dimensional fluorescence spectra were measured under the following conditions: the emission wavelength was recorded between 200 and 600 nm, the initial excitation wavelength was set at 200 nm with an increment of 5 nm, and the other scanning parameters were just the same as those for the fluorescence emission spectra. FT-IR measurements were carried out at 298 K on a Prestige-21 FT-IR spectrometer. All spectra were taken via the Attenuated Total Reflection (ATR) method with resolution of 4 cm1 and 128 scans. The FT-IR spectra of HSA (5.0 × 104 mol l1) in the presence and absence of BPAF were recorded in the range of 1450–1700 cm1. The synchronous fluorescence spectra were carried out with Δλ = 15 and 60 nm, λex = 230–350 nm. HSA concentration was kept fixed at 4.0 × 106 mol l1 in a quartz cell, and BPAF concentration ranged from 0 to 1.34 × 105 mol l1 by successive additions of 4.5 × 104 mol l1 BPAF solution (to give a final volume of 90 μl). Titrations were performed manually by using a microinjector.
RESULTS AND DISCUSSION Preparation of solutions
Fluorescence quenching mechanism
The HSA solution was prepared by dissolving HSA in Tris–HCl buffer solution (0.05 mol l1 Tris, 0.10 mol l1 NaCl, pH 7.4) and was kept in the dark below 277 K. The stock solution of BPAF was prepared by dissolving it in ultrapure water with a final concentration of 4.5 × 104 mol l1. Solutions of ketoprofen and ibuprofen were prepared by dissolving them in a small amount of ethanol, respectively, then diluting with ultrapure water to the concentration of 2.0 × 104 mol l1.
At the excitation wavelength of 280 nm, the fluorescence spectra of HSA with varying concentrations of BPAF were investigated previously (Wang et al., 2014). The fluorescence intensity of HSA decreased regularly with the increasing concentration of BPAF, but no significant shift of the emission maximum wavelength was observed, indicating that BPAF interacted with HSA and quenched its intrinsic fluorescence. Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interactions with quencher molecule, which may result from ground complex formation, energy transfer, and dynamic quenching processes (Lakowicz, 2006). The different mechanisms of fluorescence quenching are usually classified as either dynamic quenching or static quenching. Dynamic quenching refers to a process in which the fluorophore and the quencher come into contact during the lifetime of the excited state, whereas static quenching refers to fluorophore– quencher complex formation. And they can be distinguished by their different dependence on temperature (Soares et al., 2007). Dynamic quenching depends upon diffusion, because higher temperature results in larger diffusion coefficients; the bimolecular quenching constants are expected to increase with increasing temperature. In contrast, increased temperature is likely to result in decreased stability of complexes and, thus, lowers the static quenching constants. In order to confirm the type of HSA fluorescence quenching, the procedure was assumed to be dynamic quenching. Usually, the experimental data are analyzed by the well-known Stern– Volmer equation (Lakowicz, 2006):
Spectral measurements
460
The fluorescence measurements were carried out by keeping HSA concentration fixed at 4.0 × 106 mol l1 in a quartz cell, and BPAF concentration ranged from 0 to 1.34 × 105 mol l1 by successive additions of 4.5 × 104 mol l1 BPAF solution (to give a final volume of 90 μl). Six titrations were performed manually using a microinjector. An excitation wavelength of 280 nm was selected, and the emission wavelength was recorded from 300 to 500 nm, at 288, 298, and 308 K, respectively. The absorption spectra of HSA in the presence of different concentrations of BPAF were recorded in the wavelength range of 200–300 nm at 298 K, while the background (containing all system components except HSA) was subtracted. The concentration of HSA was kept at 4.0 × 106 mol l1, while that of BPAF was varied from 0 to 1.34 × 105 mol l1. Site probe competition experiments: 3 ml of BPAF–HSA mixture solution was added to a quartz cell, and the ratio of BPAF to HSA was kept at 6:1 in order to keep nonspecific binding of probes to a minimum. Then, the site probes, namely, ketoprofen and ibuprofen, were gradually added to the mixture solution, and the fluorescence spectra were recorded in the range of 300–500 nm upon excitation at 280 nm. Besides, it was confirmed that the two site probes had no absorption in the excitation and emission wavelength of HSA.
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F0 ¼ 1 þ K SV ½Q F
(1)
where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. KSV is the Stern–Volmer
Copyright © 2015 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2015; 28: 459–466
NEW INSIGHT INTO THE INTERACTIONS OF BPAF AND HSA
Binding constants and the number of binding sites For the static quenching interaction, when small molecules bind independently to a set of equivalent sites on a macromolecule, the binding constant (Ka) and the number of binding sites (n) can be determined by the following equation (Rasoulzadeh et al., 2009): F0 F lg ¼ lgK a þ nlg½Q F
(2)
where Ka and n are the binding constant and the number of binding site, respectively. By the plot of lg(F0 F)/F versus lg [Q], the values of Ka and n can be obtained and are listed in Table 1. It was found that Ka decreased with increasing temperature, resulting in a reduction of the stability of the BPAF–HSA complex. The value of n was approximately equal to 1, indicating that there was one single binding site in HSA for BPAF during their interaction. Thermodynamic parameters and nature of the binding forces There are essentially four types of noncovalent interactions that could play a role in ligands binding to proteins. They are hydrogen bond, electrostatic force, hydrophobic force, and van der
4.0
a
0.184
3.5
a
0.138
Absorbance
0.128
3.0
Absorbance
quenching constant, and [Q] is the concentration of quencher (Permyakov, 1993). KSV can be calculated from the Stern–Volmer plots of F0/F versus [Q] at three temperatures, and KSV values are listed in Table 1. The results show that the Stern–Volmer quenching constant KSV was inversely correlated with temperature, which indicated that the probable quenching mechanism was static quenching initiated by the complex formation between HSA and BPAF (Hiratsuka, 1990). The UV–vis absorption spectra of HSA in the presence and absence of BPAF were also recorded to confirm the probable quenching mechanism. As can be seen in Figure 1, there were two absorbance peaks: strong absorbance with a peak at 208 nm, which represented the content of α-helix structure of HSA, and a shoulder absorbance peak around 278 nm, which represented the n→π * transition of peptide bond and the amino acid residues (Trp, Tyr, and Phe) of HSA (Cao and Zhao, 2004). And the intensity of the two peaks changed with the addition of BPAF. As we all know, the dynamic quenching only affected the excited state of fluorophore and did not change the absorption spectrum, but the static quenching induced the change of absorption spectrum of fluorophore, so the result again confirms that the quenching mechanism was a static quenching initiated by the formation of the ground-state BPAF–HSA complex (Kandagal et al., 2006) and changes in protein conformation (Tao, 1981).
d
2.5
0.118
d
0.108 0.098 0.088
2.0
0.074
1.5
0.052 270
275
280
285
290
Wavelength/nm
1.0 0.5 0.0 200
220
240
260
280
300
320
340
Wavelength(nm) Figure 1. Ultraviolet–visible absorption spectra of human serum albumin in the absence and presence of bisphenol AF (T = 298 K). 6 1 6 1 CHSA = 4.0 × 10 mol l ; CBPAF (× 10 mol l ), (a–d) 0, 2.2, 4.6, and 6.9.
Waals interactions (Ross and Subramanian, 1981; Shohrati et al., 2007). The sign and magnitude of the thermodynamic parameters (ΔH and ΔS) are important information for confirming the main forces involved in the binding reaction. For this purpose, thermodynamic parameters were calculated from the following equation: ΔG0 ¼ RTlnK a
(3) 1
1
where R is the universal gas constant (8.314 J mol K ), T is temperature (K), and Ka is the binding constant obtained from Eq. (3). Gibbs free energy change of binding (ΔG0) was calculated using lnKa values for different temperatures. The enthalpy change (ΔH0) and entropy change (ΔS0) of binding were estimated from the following equation: lnK a ¼
ΔS0 ΔH0 R RT
(4)
According to Eq. (4), ΔH0 and ΔS0 parameters can be calculated from the slope and intercept of the plot of lnKa versus 1/T, respectively. The thermodynamic parameters are summarized in Table 1. According to the views of Ross and Subramanian (1981), if ΔH > 0 and ΔS > 0, the main binding force is hydrophobic interaction; if ΔH ≈ 0 and ΔS > 0, the main force is electrostatic interaction; if ΔH < 0 and ΔS < 0, hydrogen bond and van der Waals interactions play major roles in the binding process. As can be seen in Table 1, the negative ΔH0 and ΔS0 values indicate that the main binding forces between BPAF and HSA were hydrogen
Table 1. Quenching and binding constants and relative thermodynamic parameters of bisphenol AF–human serum albumin system at different temperatures KSV (×104 l mol1)
Ka (× 104 l mol1)
n
ΔH0 (kJ mol1)
ΔS0 (J mol1 K1)
ΔG0 (kJ mol1)
288 298 308
1.118 0.937 0.777
2.278 1.957 1.044
1.062 1.067 1.032
28.874 ± 2.11
16.112 ± 3.44
24.03 ± 1.91 24.48 ± 2.57 23.69 ± 2.09
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T (K)
L. YANG ET AL. bond and van der Waals interactions. Moreover, the negative ΔG0 shows the spontaneous nature of the binding process. Identification of the binding site HSA, a 585-residue protein, is monomeric but contains three structurally similar α-helical domains (I, II, and III); each domain has two subdomains (A and B), which are six (A) and four (B) α-helices, respectively. Drug binding sites I and II of HSA are located in hydrophobic cavities in subdomains IIA and IIIA, respectively. Several studies have shown that HSA was able to bind many ligands in several binding sites (Sugio et al., 1999; Sakurai et al., 2004; Ascenzi et al., 2005). There is a large hydrophobic cavity presenting in subdomain IIA where many drugs can bind. When two ligands (denoted with L1 and L2) bind to HSA simultaneously, two types of interaction can occur (Ni et al., 2009): (1) competitive binding þHSA
HSA. Hence, it can be concluded that BPAF was mainly bound to site I in subdomain IIA of HSA. To clearly describe the interaction between BPAF and HSA, the molecular modeling studies were also carried out (Wang et al., 2014). The results of modeling indicate that there was a large hydrophobic cavity in subdomain IIA to accommodate the BPAF, and the interaction between BPAF and HSA was hydrophobic and electrostatic synergy, which were both in accord with the earlier results. Energy transfer from HSA to BPAF According to the Förster nonradioactive resonance energy transfer theory (Forster, 1996), the distances between the protein residue (donor) and the bound drug (acceptor) in HSA can be determined. The efficiency of energy transfer between the donor and acceptor, E, can be calculated by the following equation (Horrocks and Collier, 1981): E ¼1
þL2
L1 → L1-HSA → L2-HSA-L1
þL2
R60 ¼ 8:791025 K 2 n4 ΦJ
L1 → L1-HSA → L1-HSA þ L2
Binding% ¼ F 2 =F 1 100
(5)
where F1 and F2 denote the fluorescence intensity of BPAF–HSA complex without the probe and with the probe, respectively. The plots of Bindings% against the concentration ratio of site probe to HSA are shown in Figure 2A. It was clear that the percentage of the specific site bound by BPAF decreased significantly with the addition of ketoprofen rather than ibuprofen, indicating that ketoprofen displaced BPAF from the binding site I, while ibuprofen had a little effect on the binding of BPAF to
where K2 is the orientation factor between the emission dipole of the donor and the absorption dipole of the acceptor. The dipole orientation factor, K2, is the least certain parameter in calculation of the critical transfer distance (R0). Although, theoretically, K2 can range from 0 to 4, the extreme values require very rigid orientations. If both the donor and the acceptor are tumbling rapidly and are free to assume any orientation, then K2 equals 2/3 (Horrocks and Collier, 1981). n is the refracted index of the medium, Φ is the fluorescence quantum yield of the donor, and J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the receptor. Therefore, X F ðλÞεðλÞλ4 Δλ J¼ X (8) F ðλÞΔλ where F(λ) is the fluorescence intensity of the donor in the wavelength range λ to λ+Δλ and ε(λ) is the extinction coefficient of the acceptor at λ.
800
0.030
100
(B)
(A)
b
BPAF
0.025
Absorbance
80
60
40
HSA
700 600
0.020
500
0.015
400 300
0.010
200 20
Ketoprofen
Ibuprofen
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Cprobe / C HSA
(7)
0.005 0.000 300
100
a 350
400
450
0 500
Fluorescence intensity (a.u.)
In order to identify the BPAF binding site on HSA, the site probe competition experiments were performed with two site probes, ketoprofen and ibuprofen, which specifically bind to sites I and II (Sudlow et al., 1976). After the addition of fluorescence probes, the percentage of the specific site bound by BPAF (Binding%) was determined by measuring the changes in fluorescence intensity (Sudlow et al., 1976):
Binding%
(6)
where r is the binding distance between donor and receptor and R0 is the critical distance at 50% transfer efficiency.
(2) noncompetitive binding þHSA
F R6 ¼ 6 0 6 F 0 R0 þ r
Wavelength (nm)
462
6
1
Figure 2. (A) Effect of site probes on the fluorescence of bisphenol AF (BPAF)–human serum albumin (HSA) (T = 298 K, CHSA = 4.0 × 10 mol l ). (B) Spectral overlap of ultraviolet–visible absorption spectrum of BPAF (a) with the fluorescence emission spectrum of HSA (b) (CHSA:CBPAF = 1:1, T = 298 K).
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J. Mol. Recognit. 2015; 28: 459–466
NEW INSIGHT INTO THE INTERACTIONS OF BPAF AND HSA The overlap of the absorption spectrum of BPAF and the fluorescence emission spectrum of HSA are shown in Figure 2B. In the present case, K2 = 2/3, n = 1.336, Φ = 0.118 (Lakowicz and Weber, 1973), and according to Eqs. (6)–(8), we can calculate J = 1.2 × 1016 cm3 l mol1, R0 = 1.174 nm; E = 0.0365, and r = 2.025 nm. The donor-to-acceptor distance (r) is smaller than 8 nm (Valeur and Brochon, 1999), the criterion for energy transfer phenomenon to occur, suggesting that the energy transfer between HSA and BPAF can occur with high possibility. Alterations of protein secondary structure induced by BPAF binding CD is a sensitive technique for monitoring conformational changes of protein upon interaction with small molecules. The effect of BPAF on HSA stability was also investigated by CD spectra (Figure 3). A high content of α-helixes in HSA was revealed by the two minima around 208 and 222 nm. The reasonable explanation is that the negative peaks between 208 and 209 nm, and 222 and 223 nm are both contributed to an n→π* transfer for the peptide bond within an α-helix (Moriyama and Takeda, 1999; Ahmad et al., 2006; Das et al., 2007). To ascertain the possible influence of BPAF binding on the secondary structure of HSA, CD measurement was also performed in the presence of BPAF at different concentrations. With the increasing addition of BPAF, the band intensity of curves (a–d) decreased regularly. The CD results are expressed in terms of mean residue ellipticity (MRE) in deg cm2 dmol1 according to the following equation (Chen et al., 1972): MRE ¼
CD ðmdegÞ C P nl10
(9)
where Cp is the molar concentration of the HSA, n is the number of amino acid residues, and l is the path length. The α-helix contents of free and bound HSA were calculated from MRE values at 208 nm using the following equation (Lu et al., 1987): α Helix ð%Þ ¼
MRE 208 4000 100 33 000 4000
(10)
where MRE208 is the observed MRE value at 208 nm, 4000 is
CD (mdeg)
0
-10
d -20
a 200
210
220
230
240
Wavelength (nm)
J. Mol. Recognit. 2015; 28: 459–466
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463
Figure 3. Circular dichroism (CD) spectra of the bisphenol AF–human serum albumin system (T = 298 K; CBPAF:CHSA = 0:1, 5:1, 10:1, and 20:1, from curve a to d).
the MRE of the β-form and random coil conformation cross at 208 nm and 33,000 is the MRE value of a pure α-helix at 208 nm. From Eqs. (9) and (10), the α-helicity in the secondary structure of HSA were calculated to be 53.44% in free HSA, and 52.85%, 50.15%, and 48.64% in the BPAF–HSA complex with the molar ratios of BPAF to HSA 5:1, 10:1, and 20:1, respectively. The decrease of α-helix percentage indicated that BPAF bound with the amino acid residues of main polypeptide chain of HSA and led to the destabilization of their hydrogen bonding networks (Cai et al., 2010; Zhang et al., 2012). However, the CD spectra of HSA in the absence and the presence of BPAF were similar in shape, indicating that the structure of HSA after the addition of BPAF was also predominantly α-helix. Three-dimensional fluorescence spectroscopy is a rising fluorescence analysis technique that can provide comprehensive, detailed fluorescence information of the sample, which makes the investigation of the characteristic conformational changes of protein more scientific and credible. The threedimensional fluorescence spectra and the corresponding contour diagram of HSA in the absence and presence of BPAF are shown in Figure 4, and the related characteristic parameters are presented in Table 2. As can be seen from Figure 4, Peak A was the Rayleigh scattering peak (λex = λem), and Peak B was the second-order scattering peak (λem = 2λex) (Chen et al., 1990). Peak 1 mainly revealed the spectral characteristic of tryptophan and tyrosine residues, because their intrinsic fluorescence was primarily exhibited when serum albumin was excited at 280 nm, while the fluorescence of phenylalanine residue could be negligible (Miller, 1979). Besides Peak 1, there was another visible Peak 2, which was mainly caused by the transition of P→P*of characteristic polypeptide backbone structure C¼O of HSA. The intensity of Peaks 1 and 2 changed obviously (Table 2) but to different degrees; in the absence and presence of BPAF, the fluorescence intensity ratios of Peaks 1 and 2 were 1:0.92 and 1:0.88, respectively. The decrease of the fluorescence intensity of the two peaks in combination with the CD spectral results suggested that the interaction of BPAF with HSA induced the slight unfolding of the polypeptide chain of HSA, which resulted in a conformational change of HSA to increase the exposure of some hydrophobic regions that had been buried (Chen et al., 2014). Synchronous fluorescence spectroscopy can give information about the molecular environment in the vicinity of a chromophore such as tryptophan and tyrosine, and it involves simultaneous scanning of the excitation and emission monochromators while maintaining a constant wavelength interval between them. The shift in the emission maximum (λem) reflects the changes of polarity around the chromophore molecule. When the wavelength interval (Δλ) between the excitation and emission wavelength is stabilized at 15 or 60 nm, the synchronous fluorescence gives characteristic information of tyrosine residues or tryptophan residues, respectively (Mallick et al., 2004). The synchronous fluorescence spectra at these two different wavelength intervals are presented in Figure 5. Generally, we can only see the fluorescence of tryptophan because energy transfer occurred between tyrosine and tryptophan. In this paper, as can be seen from Figure 5A, the fluorescence of tyrosine increased with addition of BPAF. This was likely due to the stretching of the peptide chains in protein
L. YANG ET AL.
Figure 4. Three-dimensional fluorescence spectra of human serum albumin (A1) and bisphenol AF–human serum albumin system (B1) and the corre-6 1 6 1 sponding contour diagrams (A2, B2). CHSA, 4.0 × 10 mol l (A1, A2, B1, B2); CBPAF, 0 (A1, A2), 7.8 × 10 mol l (B1, B2); T = 298 K.
Table 2. Three-dimensional fluorescence spectral characteristic parameters of human serum albumin (HSA) and bisphenol AF–human serum albumin (BPAF–HSA) system HSA Fluorescence peaks
Peak position λex/λem (nm nm1)
Peak 1 Peak 2
BPAF–HSA
Stokes shift Δλ (nm)
Intensity (a.u.)
Peak position λex/λem (nm nm1)
Stokes shift Δλ (nm)
Intensity (a.u.)
62.0 111.0
542.035 21.406
280.0/340.0 225.0/342.0
60.0 117.0
498.399 371.961
280.0/342.0 225.0/336.0
HSA, human serum albumin; BPAF–HSA, bisphenol AF–human serum albumin.
(B) 350
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
(A) g
300 250
a
200 150 100 50 0 240
260
280
300
Wavelength (nm)
320
340
a
800 700 600
g
500 400 300 200 100 0 240
260
280
300
320
340
Wavelength (nm)
464
Figure 5. Synchronous fluorescence spectra of bisphenol AF with human serum albumin: (A) observing the tyrosine residues at Δλ = 15 nm and (B) 6 1 6 1 observing the tryptophan residues at Δλ = 60 nm. CHSA = 4.0 × 10 mol l ; CBPAF (×10 mol l ) (a–g) 0, 2.2, 4.6, 6.7, 8.9, 11.2, and 13.4; T = 298 K.
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J. Mol. Recognit. 2015; 28: 459–466
NEW INSIGHT INTO THE INTERACTIONS OF BPAF AND HSA
(A)
(B)
1700
1650
amide II
1600
1550
1500
1450
1700
1650
1600
1537.2
amide I 1652
Absorbance
amide II 1541.1
1647.2
Absorbance
amide I
1550
1500
1450
Wavelengthnumber (cm-1)
Wavelengthnumber (cm-1)
Figure 6. Fourier-transform infrared spectroscopy spectra of free human serum albumin (A) and bisphenol AF–human serum albumin complex in Tris1 4 1 4 1 HCl buffer solution in the range of 1700 to 1450 cm (B), CBPAF, 4.5 × 10 mol l ; CHSA, 5.0 × 10 mol l .
caused by BPAF, which increased the distance between tyrosine and tryptophan, as a result, decreased the efficiency of energy transfer. Meanwhile, it is apparent from Figure 5A that the emission wavelength of the tyrosine residues was blue-shifted with increasing concentration of BPAF. This blue shift expressed that the conformation of HSA was changed, and it suggested a less polar (or more hydrophobic) environment of tyrosine residue (Jayaraman et al., 2012). As shown in Figure 5B, the fluorescence of tryptophan residues shows a very strong decrease. Besides the energy transfer discussed earlier, BPAF binding to site I (close to tryptophan) in subdomain IIA of HSA also could cause the fluorescence of tryptophan to decrease. The phenomena indicated a conformational change of HSA under the interaction with BPAF. To draw relevant conclusions on the BPAF–HSA binding mechanism, FT-IR spectroscopic measurements were performed on BPAF and the BPAF–HSA complex. In the FT-IR region, the frequencies of bands due to the amide I, II, and III vibrations are sensitive to the secondary structure of proteins. Particularly, the amide I band is useful for the secondary structure studies. The protein amide I band 1600–1700 cm1 (mainly C¼O stretch) and amide II band 1541 cm1 (C–N stretch coupled with N–H bending mode) both have a relationship with the secondary structure of protein (Rahmelow and Hubner, 1996). However, the amide I band is more sensitive to the change of protein secondary structure than amide II (Witold et al., 1993; Wi et al., 1998). Figures 6A and 6B respectively show FT-IR spectra of free HSA and BPAF–HSA complex in Tris-HCl buffer solution in the range of 1700 to 1450 cm1. The peak position of amide I moved from 1647.2 to 1652 cm1, and amide II moved from 1541.1 to 1537.2 cm1 in HSA infrared spectrum after interaction with BPAF, which indicates that the secondary structure of HSA has been changed because of the interaction of BPAF with HSA.
According to all the phenomena and analyses earlier, it can be concluded that the binding of BPAF to HSA induced some conformational changes in HSA.
CONCLUSIONS In this paper, the interaction between BPAF and HSA has been investigated by multispectroscopic techniques. BPAF effectively quenched the fluorescence of HSA by a static quenching process. Based on the results of binding capacity and calculated thermodynamic parameters, it can be concluded that BPAF can spontaneously bind with HSA through hydrogen bond and van der Waals interactions. The site-competitive replacement experiments showed that BPAF bound to HSA on the subdomain IIA. CD, FT-IR, three-dimensional fluorescence spectra, and synchronous fluorescence spectroscopy revealed that the microenvironment and conformation of HSA were demonstrably changed in the presence of BPAF. All these experimental results and theoretical data demonstrate that BPAF has an obvious denaturing effect on HSA. This study is expected to provide an important insight into the interactions of the important physiological protein with endocrine disruptors–BPAF and a theoretical basis for ecotoxicology and environmental risk assessment.
Acknowledgements This project was supported by the National Natural Science Foundation China (Grant No. 51178212); Natural Science Foundation of Liaoning Province, China (No. 201102082); and Foundation of 211Project for Innovative Talents Training, Liaoning University and the Foundation for Young Scholars of Liaoning University. The authors also thank our colleagues and other students who participated in this work.
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J. Mol. Recognit. 2015; 28: 459–466
Revised: 29 October 2014,
Accepted: 14 January 2015,
Published online in Wiley Online Library: 19 February 2015
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2463
Direct interactions in the recognition between the environmental estrogen bisphenol AF and human serum albumin Lijun Yang, Junna Lv, Xin Wang, Jing Zhang, Qi Li, Tingting Zhang, Zhenzhen Zhang and Lei Zhang* Bisphenol AF (BPAF) was used as a model compound to investigate the binding mechanism between the endocrine disrupting compound and human serum albumin (HSA) using multispectroscopic techniques and molecular modeling method at the protein level. The results indicated that BPAF was indeed bound to HSA and located in the hydrophobic pocket of HSA on subdomain IIA through hydrogen bond and van der Waals interactions. The fluorescence quenching data showed that the binding of BPAF and HSA quenched the intrinsic fluorescence of HSA, and the static quenching constants were acquired. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: Bisphenol AF; Human serum albumin; Interaction; Multi-spectroscopic techniques
INTRODUCTION
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* Correspondence to: Lei Zhang, College of Chemistry, Liaoning University, Shenyang 110036, China. E-mail: [email protected] L. Yang, J. Lv, X. Wang, J. Zhang, Q. Li, T. Zhang, Z. Zhang, L. Zhang College of Chemistry, Liaoning University, Shenyang 110036, China
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It is well known that endocrine disruptors attract the interests of an increasing number of scientists because of their possible negative effects on human health. Some endocrine disruptors have been reported to be environmental pollutants that can give rise to abnormal sexual development and abnormal feminizing on animals (Ohko et al., 2001). Among these endocrine disruptors, bisphenol A (BPA) has been given special attention (Rochester, 2013; Pan et al., 2014). Bisphenol AF [1,1,1,3,3,3-hexafluoro-2,2-bis(4-hydroxyphenyl)propane] (BPAF), the fluorinated homologue of BPA, has CF3 moiety, which may be much more electronegative and reactive than the CH3 of BPA. Therefore, BPAF may pose high potentiality as an endocrine disruptor for humans and wildlife via binding with hormone receptors (Kitamura et al., 2005). BPAF has broad applications in the areas such as electronic devices, optical fibers, and food processing equipment (Akahori et al., 2008; LaFleur and Schug, 2011). It has become one of the high-yielding chemicals in the world because of its growing demand. Because of its wide usage in the environment and its estrogenic activity in specific responses in vitro and in vivo, it is reasonable to assume that BPAF may be harmful to human health. Binding of xenobiotics to plasma proteins has toxicological importance, because it alters their free, active concentrations, and as a consequence, the degree and time of action in the body also affects the duration and intensity of their effects (Kragh-Hansen, 1981; Cserháti and Forgcás, 1995). BPAF’s effects, however, on serum albumin remain unclear. The affinity between BPAF and serum albumin is an important factor to understand the toxicological properties of BPAF as it strongly influences BPAF distribution and determines the free fraction that is available for subsequent interactions with targeted receptors.
Serum albumin, the most abundant protein in plasma, functions in the maintenance of colloid osmotic blood pressure and in the binding and transportation of various ligands such as fatty acids, hormones, and drugs (Zolese et al., 2000). The distribution, free concentration, and metabolism of these ligands strongly depend on their binding constants with serum albumin (Wu et al., 2007). Human serum albumin (HSA) is an ideal protein to use in studies of endocrine disruptor–protein binding (Westermark et al., 1987; Engel et al., 2008). Therefore, investigating the interaction of BPAF and HSA is significant for understanding its transport and distribution in the body and for clarifying its action mechanisms and pharmacodynamics. As of yet, however, no work has been reported for the mechanism of this interaction and the detailed physicochemical characterizations of BPAF binding to HSA. In this paper, the interaction between BPAF and HSA was systematically studied by multispectroscopic techniques. We estimated the association constants, number of binding sites, thermodynamic parameters, and binding force for the interaction of BPAF with HSA. The specific binding site of BPAF on HSA was investigated in detail. The effect of BPAF on the microenvironment and conformation of HSA was also studied. This study provides basic data for clarifying the binding mechanisms of BPAF with HSA and is helpful for understanding its effect on protein function during its transportation in the blood and its toxicity in vivo.
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EXPERIMENTAL Reagents and apparatus HSA (CAS#70024-90-7) (purity > 99%) was purchased from Heowns Biochemical Technology Co., Ltd. (Tianjin, China). The analytical standard BPAF (CAS#1478-61-1) (chemical reference substance, 97.0% purity) was purchased from Heowns Biochemical Technology Co., Ltd. (Tianjin, China). Ketoprofen and ibuprofen were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). All other chemicals were of analytical reagent grade. Ultrapure water used throughout experiments was purified using a Sartorius Arium 611 system (Sartorius AG, Göttingen, Germany). All fluorescence measurements were carried out through a Cary Eclipse fluorescence spectrophotometer (Varian Inc., Palo Alto, CA, USA) equipped with a thermostat bath and a 1-cm quartz cell. The absorption spectra were recorded on the ultraviolet–visible (UV–Vis) Cary 5000 spectrophotometer (Varian Inc., Palo Alto, CA, USA) equipped with two 1-cm quartz cells. Circular dichroism (CD) measurements were performed on a J-810 spectro-polarimeter (JASCO Corp., Tokyo, Japan) using a 1-cm quartz cell. Fourier-transform infrared spectroscopy (FT-IR) measurements were carried out at room temperature on a Prestige-21 FT-IR spectrometer (SHIMADZU Inc., Kyoto, Japan).
The CD spectra of HSA (4.0 × 107 mol l1) in the presence of BPAF were recorded in the range of 200–250 nm at 298 K under constant nitrogen flush. The molar ratio of BPAF to HSA was varied as 0:1, 5:1, 10:1, and 20:1, and each CD spectrum was the average of three successive scans. Three-dimensional fluorescence spectra were measured under the following conditions: the emission wavelength was recorded between 200 and 600 nm, the initial excitation wavelength was set at 200 nm with an increment of 5 nm, and the other scanning parameters were just the same as those for the fluorescence emission spectra. FT-IR measurements were carried out at 298 K on a Prestige-21 FT-IR spectrometer. All spectra were taken via the Attenuated Total Reflection (ATR) method with resolution of 4 cm1 and 128 scans. The FT-IR spectra of HSA (5.0 × 104 mol l1) in the presence and absence of BPAF were recorded in the range of 1450–1700 cm1. The synchronous fluorescence spectra were carried out with Δλ = 15 and 60 nm, λex = 230–350 nm. HSA concentration was kept fixed at 4.0 × 106 mol l1 in a quartz cell, and BPAF concentration ranged from 0 to 1.34 × 105 mol l1 by successive additions of 4.5 × 104 mol l1 BPAF solution (to give a final volume of 90 μl). Titrations were performed manually by using a microinjector.
RESULTS AND DISCUSSION Preparation of solutions
Fluorescence quenching mechanism
The HSA solution was prepared by dissolving HSA in Tris–HCl buffer solution (0.05 mol l1 Tris, 0.10 mol l1 NaCl, pH 7.4) and was kept in the dark below 277 K. The stock solution of BPAF was prepared by dissolving it in ultrapure water with a final concentration of 4.5 × 104 mol l1. Solutions of ketoprofen and ibuprofen were prepared by dissolving them in a small amount of ethanol, respectively, then diluting with ultrapure water to the concentration of 2.0 × 104 mol l1.
At the excitation wavelength of 280 nm, the fluorescence spectra of HSA with varying concentrations of BPAF were investigated previously (Wang et al., 2014). The fluorescence intensity of HSA decreased regularly with the increasing concentration of BPAF, but no significant shift of the emission maximum wavelength was observed, indicating that BPAF interacted with HSA and quenched its intrinsic fluorescence. Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interactions with quencher molecule, which may result from ground complex formation, energy transfer, and dynamic quenching processes (Lakowicz, 2006). The different mechanisms of fluorescence quenching are usually classified as either dynamic quenching or static quenching. Dynamic quenching refers to a process in which the fluorophore and the quencher come into contact during the lifetime of the excited state, whereas static quenching refers to fluorophore– quencher complex formation. And they can be distinguished by their different dependence on temperature (Soares et al., 2007). Dynamic quenching depends upon diffusion, because higher temperature results in larger diffusion coefficients; the bimolecular quenching constants are expected to increase with increasing temperature. In contrast, increased temperature is likely to result in decreased stability of complexes and, thus, lowers the static quenching constants. In order to confirm the type of HSA fluorescence quenching, the procedure was assumed to be dynamic quenching. Usually, the experimental data are analyzed by the well-known Stern– Volmer equation (Lakowicz, 2006):
Spectral measurements
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The fluorescence measurements were carried out by keeping HSA concentration fixed at 4.0 × 106 mol l1 in a quartz cell, and BPAF concentration ranged from 0 to 1.34 × 105 mol l1 by successive additions of 4.5 × 104 mol l1 BPAF solution (to give a final volume of 90 μl). Six titrations were performed manually using a microinjector. An excitation wavelength of 280 nm was selected, and the emission wavelength was recorded from 300 to 500 nm, at 288, 298, and 308 K, respectively. The absorption spectra of HSA in the presence of different concentrations of BPAF were recorded in the wavelength range of 200–300 nm at 298 K, while the background (containing all system components except HSA) was subtracted. The concentration of HSA was kept at 4.0 × 106 mol l1, while that of BPAF was varied from 0 to 1.34 × 105 mol l1. Site probe competition experiments: 3 ml of BPAF–HSA mixture solution was added to a quartz cell, and the ratio of BPAF to HSA was kept at 6:1 in order to keep nonspecific binding of probes to a minimum. Then, the site probes, namely, ketoprofen and ibuprofen, were gradually added to the mixture solution, and the fluorescence spectra were recorded in the range of 300–500 nm upon excitation at 280 nm. Besides, it was confirmed that the two site probes had no absorption in the excitation and emission wavelength of HSA.
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F0 ¼ 1 þ K SV ½Q F
(1)
where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. KSV is the Stern–Volmer
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NEW INSIGHT INTO THE INTERACTIONS OF BPAF AND HSA
Binding constants and the number of binding sites For the static quenching interaction, when small molecules bind independently to a set of equivalent sites on a macromolecule, the binding constant (Ka) and the number of binding sites (n) can be determined by the following equation (Rasoulzadeh et al., 2009): F0 F lg ¼ lgK a þ nlg½Q F
(2)
where Ka and n are the binding constant and the number of binding site, respectively. By the plot of lg(F0 F)/F versus lg [Q], the values of Ka and n can be obtained and are listed in Table 1. It was found that Ka decreased with increasing temperature, resulting in a reduction of the stability of the BPAF–HSA complex. The value of n was approximately equal to 1, indicating that there was one single binding site in HSA for BPAF during their interaction. Thermodynamic parameters and nature of the binding forces There are essentially four types of noncovalent interactions that could play a role in ligands binding to proteins. They are hydrogen bond, electrostatic force, hydrophobic force, and van der
4.0
a
0.184
3.5
a
0.138
Absorbance
0.128
3.0
Absorbance
quenching constant, and [Q] is the concentration of quencher (Permyakov, 1993). KSV can be calculated from the Stern–Volmer plots of F0/F versus [Q] at three temperatures, and KSV values are listed in Table 1. The results show that the Stern–Volmer quenching constant KSV was inversely correlated with temperature, which indicated that the probable quenching mechanism was static quenching initiated by the complex formation between HSA and BPAF (Hiratsuka, 1990). The UV–vis absorption spectra of HSA in the presence and absence of BPAF were also recorded to confirm the probable quenching mechanism. As can be seen in Figure 1, there were two absorbance peaks: strong absorbance with a peak at 208 nm, which represented the content of α-helix structure of HSA, and a shoulder absorbance peak around 278 nm, which represented the n→π * transition of peptide bond and the amino acid residues (Trp, Tyr, and Phe) of HSA (Cao and Zhao, 2004). And the intensity of the two peaks changed with the addition of BPAF. As we all know, the dynamic quenching only affected the excited state of fluorophore and did not change the absorption spectrum, but the static quenching induced the change of absorption spectrum of fluorophore, so the result again confirms that the quenching mechanism was a static quenching initiated by the formation of the ground-state BPAF–HSA complex (Kandagal et al., 2006) and changes in protein conformation (Tao, 1981).
d
2.5
0.118
d
0.108 0.098 0.088
2.0
0.074
1.5
0.052 270
275
280
285
290
Wavelength/nm
1.0 0.5 0.0 200
220
240
260
280
300
320
340
Wavelength(nm) Figure 1. Ultraviolet–visible absorption spectra of human serum albumin in the absence and presence of bisphenol AF (T = 298 K). 6 1 6 1 CHSA = 4.0 × 10 mol l ; CBPAF (× 10 mol l ), (a–d) 0, 2.2, 4.6, and 6.9.
Waals interactions (Ross and Subramanian, 1981; Shohrati et al., 2007). The sign and magnitude of the thermodynamic parameters (ΔH and ΔS) are important information for confirming the main forces involved in the binding reaction. For this purpose, thermodynamic parameters were calculated from the following equation: ΔG0 ¼ RTlnK a
(3) 1
1
where R is the universal gas constant (8.314 J mol K ), T is temperature (K), and Ka is the binding constant obtained from Eq. (3). Gibbs free energy change of binding (ΔG0) was calculated using lnKa values for different temperatures. The enthalpy change (ΔH0) and entropy change (ΔS0) of binding were estimated from the following equation: lnK a ¼
ΔS0 ΔH0 R RT
(4)
According to Eq. (4), ΔH0 and ΔS0 parameters can be calculated from the slope and intercept of the plot of lnKa versus 1/T, respectively. The thermodynamic parameters are summarized in Table 1. According to the views of Ross and Subramanian (1981), if ΔH > 0 and ΔS > 0, the main binding force is hydrophobic interaction; if ΔH ≈ 0 and ΔS > 0, the main force is electrostatic interaction; if ΔH < 0 and ΔS < 0, hydrogen bond and van der Waals interactions play major roles in the binding process. As can be seen in Table 1, the negative ΔH0 and ΔS0 values indicate that the main binding forces between BPAF and HSA were hydrogen
Table 1. Quenching and binding constants and relative thermodynamic parameters of bisphenol AF–human serum albumin system at different temperatures KSV (×104 l mol1)
Ka (× 104 l mol1)
n
ΔH0 (kJ mol1)
ΔS0 (J mol1 K1)
ΔG0 (kJ mol1)
288 298 308
1.118 0.937 0.777
2.278 1.957 1.044
1.062 1.067 1.032
28.874 ± 2.11
16.112 ± 3.44
24.03 ± 1.91 24.48 ± 2.57 23.69 ± 2.09
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T (K)
L. YANG ET AL. bond and van der Waals interactions. Moreover, the negative ΔG0 shows the spontaneous nature of the binding process. Identification of the binding site HSA, a 585-residue protein, is monomeric but contains three structurally similar α-helical domains (I, II, and III); each domain has two subdomains (A and B), which are six (A) and four (B) α-helices, respectively. Drug binding sites I and II of HSA are located in hydrophobic cavities in subdomains IIA and IIIA, respectively. Several studies have shown that HSA was able to bind many ligands in several binding sites (Sugio et al., 1999; Sakurai et al., 2004; Ascenzi et al., 2005). There is a large hydrophobic cavity presenting in subdomain IIA where many drugs can bind. When two ligands (denoted with L1 and L2) bind to HSA simultaneously, two types of interaction can occur (Ni et al., 2009): (1) competitive binding þHSA
HSA. Hence, it can be concluded that BPAF was mainly bound to site I in subdomain IIA of HSA. To clearly describe the interaction between BPAF and HSA, the molecular modeling studies were also carried out (Wang et al., 2014). The results of modeling indicate that there was a large hydrophobic cavity in subdomain IIA to accommodate the BPAF, and the interaction between BPAF and HSA was hydrophobic and electrostatic synergy, which were both in accord with the earlier results. Energy transfer from HSA to BPAF According to the Förster nonradioactive resonance energy transfer theory (Forster, 1996), the distances between the protein residue (donor) and the bound drug (acceptor) in HSA can be determined. The efficiency of energy transfer between the donor and acceptor, E, can be calculated by the following equation (Horrocks and Collier, 1981): E ¼1
þL2
L1 → L1-HSA → L2-HSA-L1
þL2
R60 ¼ 8:791025 K 2 n4 ΦJ
L1 → L1-HSA → L1-HSA þ L2
Binding% ¼ F 2 =F 1 100
(5)
where F1 and F2 denote the fluorescence intensity of BPAF–HSA complex without the probe and with the probe, respectively. The plots of Bindings% against the concentration ratio of site probe to HSA are shown in Figure 2A. It was clear that the percentage of the specific site bound by BPAF decreased significantly with the addition of ketoprofen rather than ibuprofen, indicating that ketoprofen displaced BPAF from the binding site I, while ibuprofen had a little effect on the binding of BPAF to
where K2 is the orientation factor between the emission dipole of the donor and the absorption dipole of the acceptor. The dipole orientation factor, K2, is the least certain parameter in calculation of the critical transfer distance (R0). Although, theoretically, K2 can range from 0 to 4, the extreme values require very rigid orientations. If both the donor and the acceptor are tumbling rapidly and are free to assume any orientation, then K2 equals 2/3 (Horrocks and Collier, 1981). n is the refracted index of the medium, Φ is the fluorescence quantum yield of the donor, and J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the receptor. Therefore, X F ðλÞεðλÞλ4 Δλ J¼ X (8) F ðλÞΔλ where F(λ) is the fluorescence intensity of the donor in the wavelength range λ to λ+Δλ and ε(λ) is the extinction coefficient of the acceptor at λ.
800
0.030
100
(B)
(A)
b
BPAF
0.025
Absorbance
80
60
40
HSA
700 600
0.020
500
0.015
400 300
0.010
200 20
Ketoprofen
Ibuprofen
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Cprobe / C HSA
(7)
0.005 0.000 300
100
a 350
400
450
0 500
Fluorescence intensity (a.u.)
In order to identify the BPAF binding site on HSA, the site probe competition experiments were performed with two site probes, ketoprofen and ibuprofen, which specifically bind to sites I and II (Sudlow et al., 1976). After the addition of fluorescence probes, the percentage of the specific site bound by BPAF (Binding%) was determined by measuring the changes in fluorescence intensity (Sudlow et al., 1976):
Binding%
(6)
where r is the binding distance between donor and receptor and R0 is the critical distance at 50% transfer efficiency.
(2) noncompetitive binding þHSA
F R6 ¼ 6 0 6 F 0 R0 þ r
Wavelength (nm)
462
6
1
Figure 2. (A) Effect of site probes on the fluorescence of bisphenol AF (BPAF)–human serum albumin (HSA) (T = 298 K, CHSA = 4.0 × 10 mol l ). (B) Spectral overlap of ultraviolet–visible absorption spectrum of BPAF (a) with the fluorescence emission spectrum of HSA (b) (CHSA:CBPAF = 1:1, T = 298 K).
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NEW INSIGHT INTO THE INTERACTIONS OF BPAF AND HSA The overlap of the absorption spectrum of BPAF and the fluorescence emission spectrum of HSA are shown in Figure 2B. In the present case, K2 = 2/3, n = 1.336, Φ = 0.118 (Lakowicz and Weber, 1973), and according to Eqs. (6)–(8), we can calculate J = 1.2 × 1016 cm3 l mol1, R0 = 1.174 nm; E = 0.0365, and r = 2.025 nm. The donor-to-acceptor distance (r) is smaller than 8 nm (Valeur and Brochon, 1999), the criterion for energy transfer phenomenon to occur, suggesting that the energy transfer between HSA and BPAF can occur with high possibility. Alterations of protein secondary structure induced by BPAF binding CD is a sensitive technique for monitoring conformational changes of protein upon interaction with small molecules. The effect of BPAF on HSA stability was also investigated by CD spectra (Figure 3). A high content of α-helixes in HSA was revealed by the two minima around 208 and 222 nm. The reasonable explanation is that the negative peaks between 208 and 209 nm, and 222 and 223 nm are both contributed to an n→π* transfer for the peptide bond within an α-helix (Moriyama and Takeda, 1999; Ahmad et al., 2006; Das et al., 2007). To ascertain the possible influence of BPAF binding on the secondary structure of HSA, CD measurement was also performed in the presence of BPAF at different concentrations. With the increasing addition of BPAF, the band intensity of curves (a–d) decreased regularly. The CD results are expressed in terms of mean residue ellipticity (MRE) in deg cm2 dmol1 according to the following equation (Chen et al., 1972): MRE ¼
CD ðmdegÞ C P nl10
(9)
where Cp is the molar concentration of the HSA, n is the number of amino acid residues, and l is the path length. The α-helix contents of free and bound HSA were calculated from MRE values at 208 nm using the following equation (Lu et al., 1987): α Helix ð%Þ ¼
MRE 208 4000 100 33 000 4000
(10)
where MRE208 is the observed MRE value at 208 nm, 4000 is
CD (mdeg)
0
-10
d -20
a 200
210
220
230
240
Wavelength (nm)
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Figure 3. Circular dichroism (CD) spectra of the bisphenol AF–human serum albumin system (T = 298 K; CBPAF:CHSA = 0:1, 5:1, 10:1, and 20:1, from curve a to d).
the MRE of the β-form and random coil conformation cross at 208 nm and 33,000 is the MRE value of a pure α-helix at 208 nm. From Eqs. (9) and (10), the α-helicity in the secondary structure of HSA were calculated to be 53.44% in free HSA, and 52.85%, 50.15%, and 48.64% in the BPAF–HSA complex with the molar ratios of BPAF to HSA 5:1, 10:1, and 20:1, respectively. The decrease of α-helix percentage indicated that BPAF bound with the amino acid residues of main polypeptide chain of HSA and led to the destabilization of their hydrogen bonding networks (Cai et al., 2010; Zhang et al., 2012). However, the CD spectra of HSA in the absence and the presence of BPAF were similar in shape, indicating that the structure of HSA after the addition of BPAF was also predominantly α-helix. Three-dimensional fluorescence spectroscopy is a rising fluorescence analysis technique that can provide comprehensive, detailed fluorescence information of the sample, which makes the investigation of the characteristic conformational changes of protein more scientific and credible. The threedimensional fluorescence spectra and the corresponding contour diagram of HSA in the absence and presence of BPAF are shown in Figure 4, and the related characteristic parameters are presented in Table 2. As can be seen from Figure 4, Peak A was the Rayleigh scattering peak (λex = λem), and Peak B was the second-order scattering peak (λem = 2λex) (Chen et al., 1990). Peak 1 mainly revealed the spectral characteristic of tryptophan and tyrosine residues, because their intrinsic fluorescence was primarily exhibited when serum albumin was excited at 280 nm, while the fluorescence of phenylalanine residue could be negligible (Miller, 1979). Besides Peak 1, there was another visible Peak 2, which was mainly caused by the transition of P→P*of characteristic polypeptide backbone structure C¼O of HSA. The intensity of Peaks 1 and 2 changed obviously (Table 2) but to different degrees; in the absence and presence of BPAF, the fluorescence intensity ratios of Peaks 1 and 2 were 1:0.92 and 1:0.88, respectively. The decrease of the fluorescence intensity of the two peaks in combination with the CD spectral results suggested that the interaction of BPAF with HSA induced the slight unfolding of the polypeptide chain of HSA, which resulted in a conformational change of HSA to increase the exposure of some hydrophobic regions that had been buried (Chen et al., 2014). Synchronous fluorescence spectroscopy can give information about the molecular environment in the vicinity of a chromophore such as tryptophan and tyrosine, and it involves simultaneous scanning of the excitation and emission monochromators while maintaining a constant wavelength interval between them. The shift in the emission maximum (λem) reflects the changes of polarity around the chromophore molecule. When the wavelength interval (Δλ) between the excitation and emission wavelength is stabilized at 15 or 60 nm, the synchronous fluorescence gives characteristic information of tyrosine residues or tryptophan residues, respectively (Mallick et al., 2004). The synchronous fluorescence spectra at these two different wavelength intervals are presented in Figure 5. Generally, we can only see the fluorescence of tryptophan because energy transfer occurred between tyrosine and tryptophan. In this paper, as can be seen from Figure 5A, the fluorescence of tyrosine increased with addition of BPAF. This was likely due to the stretching of the peptide chains in protein
L. YANG ET AL.
Figure 4. Three-dimensional fluorescence spectra of human serum albumin (A1) and bisphenol AF–human serum albumin system (B1) and the corre-6 1 6 1 sponding contour diagrams (A2, B2). CHSA, 4.0 × 10 mol l (A1, A2, B1, B2); CBPAF, 0 (A1, A2), 7.8 × 10 mol l (B1, B2); T = 298 K.
Table 2. Three-dimensional fluorescence spectral characteristic parameters of human serum albumin (HSA) and bisphenol AF–human serum albumin (BPAF–HSA) system HSA Fluorescence peaks
Peak position λex/λem (nm nm1)
Peak 1 Peak 2
BPAF–HSA
Stokes shift Δλ (nm)
Intensity (a.u.)
Peak position λex/λem (nm nm1)
Stokes shift Δλ (nm)
Intensity (a.u.)
62.0 111.0
542.035 21.406
280.0/340.0 225.0/342.0
60.0 117.0
498.399 371.961
280.0/342.0 225.0/336.0
HSA, human serum albumin; BPAF–HSA, bisphenol AF–human serum albumin.
(B) 350
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
(A) g
300 250
a
200 150 100 50 0 240
260
280
300
Wavelength (nm)
320
340
a
800 700 600
g
500 400 300 200 100 0 240
260
280
300
320
340
Wavelength (nm)
464
Figure 5. Synchronous fluorescence spectra of bisphenol AF with human serum albumin: (A) observing the tyrosine residues at Δλ = 15 nm and (B) 6 1 6 1 observing the tryptophan residues at Δλ = 60 nm. CHSA = 4.0 × 10 mol l ; CBPAF (×10 mol l ) (a–g) 0, 2.2, 4.6, 6.7, 8.9, 11.2, and 13.4; T = 298 K.
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NEW INSIGHT INTO THE INTERACTIONS OF BPAF AND HSA
(A)
(B)
1700
1650
amide II
1600
1550
1500
1450
1700
1650
1600
1537.2
amide I 1652
Absorbance
amide II 1541.1
1647.2
Absorbance
amide I
1550
1500
1450
Wavelengthnumber (cm-1)
Wavelengthnumber (cm-1)
Figure 6. Fourier-transform infrared spectroscopy spectra of free human serum albumin (A) and bisphenol AF–human serum albumin complex in Tris1 4 1 4 1 HCl buffer solution in the range of 1700 to 1450 cm (B), CBPAF, 4.5 × 10 mol l ; CHSA, 5.0 × 10 mol l .
caused by BPAF, which increased the distance between tyrosine and tryptophan, as a result, decreased the efficiency of energy transfer. Meanwhile, it is apparent from Figure 5A that the emission wavelength of the tyrosine residues was blue-shifted with increasing concentration of BPAF. This blue shift expressed that the conformation of HSA was changed, and it suggested a less polar (or more hydrophobic) environment of tyrosine residue (Jayaraman et al., 2012). As shown in Figure 5B, the fluorescence of tryptophan residues shows a very strong decrease. Besides the energy transfer discussed earlier, BPAF binding to site I (close to tryptophan) in subdomain IIA of HSA also could cause the fluorescence of tryptophan to decrease. The phenomena indicated a conformational change of HSA under the interaction with BPAF. To draw relevant conclusions on the BPAF–HSA binding mechanism, FT-IR spectroscopic measurements were performed on BPAF and the BPAF–HSA complex. In the FT-IR region, the frequencies of bands due to the amide I, II, and III vibrations are sensitive to the secondary structure of proteins. Particularly, the amide I band is useful for the secondary structure studies. The protein amide I band 1600–1700 cm1 (mainly C¼O stretch) and amide II band 1541 cm1 (C–N stretch coupled with N–H bending mode) both have a relationship with the secondary structure of protein (Rahmelow and Hubner, 1996). However, the amide I band is more sensitive to the change of protein secondary structure than amide II (Witold et al., 1993; Wi et al., 1998). Figures 6A and 6B respectively show FT-IR spectra of free HSA and BPAF–HSA complex in Tris-HCl buffer solution in the range of 1700 to 1450 cm1. The peak position of amide I moved from 1647.2 to 1652 cm1, and amide II moved from 1541.1 to 1537.2 cm1 in HSA infrared spectrum after interaction with BPAF, which indicates that the secondary structure of HSA has been changed because of the interaction of BPAF with HSA.
According to all the phenomena and analyses earlier, it can be concluded that the binding of BPAF to HSA induced some conformational changes in HSA.
CONCLUSIONS In this paper, the interaction between BPAF and HSA has been investigated by multispectroscopic techniques. BPAF effectively quenched the fluorescence of HSA by a static quenching process. Based on the results of binding capacity and calculated thermodynamic parameters, it can be concluded that BPAF can spontaneously bind with HSA through hydrogen bond and van der Waals interactions. The site-competitive replacement experiments showed that BPAF bound to HSA on the subdomain IIA. CD, FT-IR, three-dimensional fluorescence spectra, and synchronous fluorescence spectroscopy revealed that the microenvironment and conformation of HSA were demonstrably changed in the presence of BPAF. All these experimental results and theoretical data demonstrate that BPAF has an obvious denaturing effect on HSA. This study is expected to provide an important insight into the interactions of the important physiological protein with endocrine disruptors–BPAF and a theoretical basis for ecotoxicology and environmental risk assessment.
Acknowledgements This project was supported by the National Natural Science Foundation China (Grant No. 51178212); Natural Science Foundation of Liaoning Province, China (No. 201102082); and Foundation of 211Project for Innovative Talents Training, Liaoning University and the Foundation for Young Scholars of Liaoning University. The authors also thank our colleagues and other students who participated in this work.
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