Article pubs.acs.org/JPCB
Exploring the Interaction of Bisphenol‑S with Serum Albumins: A Better or Worse Alternative for Bisphenol A? Manjumol Mathew,† S. Sreedhanya,‡ P. Manoj,§ C. T. Aravindakumar,§,∥ and Usha K. Aravind*,†,⊥ †
Advanced Centre of Environmental Studies and Sustainable Development, ‡School of Chemical Sciences, §Inter-University Instrumentation Centre, ∥School of Environmental Sciences, Mahatma Gandhi University, Kottayam-686 560, India ⊥ Centre for Environment Education and Technology (CEET), Kottayam, Kerala-686 560, India S Supporting Information *
ABSTRACT: The interaction of bisphenol-S (BPS) with serum albumins using steady-state, synchronous, timeresolved, and circular dichroism spectroscopies has been investigated. The binding interactions have also been investigated in the case of bisphenol A (BPA). The fluorescence quenching pathways are different for both of these endocrine disrupting compounds. Steady-state and timeresolved studies reveal static quenching at lower concentrations of BPS and dynamic quenching at higher concentrations. CD results also maintained the concentration dependent variation with a complete distortion of α-helices at 10−5 M BPS. Besides this, addition of sodium dodecyl sulfate (SDS) results in the further unfolding of protein in the case of BPS, whereas time-resolved studies indicated refolding for BPA denatured human serum albumin (HSA). The entire study indicates an irreversible binding of BPS with HSA. Hence, these results reveal the possible involvement of BPS in the physiological pathway raising a health threat as already their presences in body fluids are known.
1. INTRODUCTION The existence of endocrine disrupting compounds (EDCs), known hormone mimicking chemicals, are not all desired in any of the environmental matrixes. However, to the contrary, their widespread usage as pesticides, industrial chemicals, plastics, plasticizers, and fuels has marked their presence everywhere, including human secretions. Bisphenols, with wide application potential, are also established EDCs. Primarily, BPA was widely applied in the production of epoxy resins, polycarbonate plastics, food cans, thermal printer paper, and dental composites/sealants.1 BPA is known to leach from these polymers, paving its way to the human body through dermal exposure and dietary intake.1c,2 The health impacts of BPA have been implicated in many in vivo and in vitro studies.3 A number of countries banned the usage of products suspected to contain BPA. The efforts were also to replace BPA with its analogues bisphenol B (BPB), bisphenol F (BPF), bisphenol S (BPS), and bisphenol AF (BPAF). Soon these alternatives marked their presence in the human body, showing a negative health impact. BPS is perhaps one of the analogues, extensively applied in the place of BPA. Now the presence of BPS can be expected in almost all consumer goods where BPA was initially in use. Similar to BPA, dermal, dust ingestion, and dietary exposures are the main pathways to the human body.4 The presence of BPS at a concentration range of several nanograms per gram is already found in canned food stuffs.5 One of the major industries that replaced BPA due to its high occurrence (∼3− © 2014 American Chemical Society
22 g/kg) is thermal paper. And the thermal paper carries BPS to all recycled paper products, making the dermal exposure inevitable. BPA analogues BPS, BPF, and others have similar estrogenic activity and even more environmental persistence. A high concentration (nanogram/gram to milligram/gram) of BPS is reported to be present in thermal receipt papers, indoor dust, and recycled paper products collected from cities in USA, Japan, Korea, and Vietnam.4b Such findings point out the possibility of occupational hazards and possibility of high levels of daily exposure. Studies of Chunyang Liao et al. indicate the occurrence of BPS in human urine samples from the United States and Seven Asian Countries.6 Low doses of this chemical are also found to disrupt nongenomic signaling pathways in cultured pituitary cells.7 The compounds’ relative inability to biodegrade can lead to its bioaccumulation and likely persistence in the environment.8 Once such toxic compound enters the body, their fate (transport and delivery) is mainly determined by human serum albumin (HSA), the most prominent carrier protein in the circulatory system.9 This possible protein−pollutant binding can cause alteration in the protein structure and can hence get in the way of its normal functioning. Received: January 14, 2014 Revised: March 10, 2014 Published: March 17, 2014 3832
dx.doi.org/10.1021/jp500404u | J. Phys. Chem. B 2014, 118, 3832−3843
The Journal of Physical Chemistry B
Article
without further purification. Buffer solution (pH 7.4) consisted of 0.05 M Tris−HCl (SRL, India). Protein solutions (1 × 10−5 M) were prepared in buffer. BPS was prepared in an ethanol− water mixture and BPA in ethanol. Ultrapure water (Millipore, Milli-Q Lab) was used throughout for experiments. All other reagents were of analytical grade. 2.2. Steady-State Measurements. The fluorescence measurements were carried out on an LS55 instrument (Perkin-Elmer) having a 20 kW continuous powered high pressure Xe lamp as the excitation source and an R928 photomultiplier as the photodetector. The excitation and emission slits were set at 5 nm. A 10 μM protein solution was used to record the spectra in a 50 mM buffer solution (pH 7.4) with varying concentrations of BPS at 298 K. Serum albumins were excited at 295 nm in order to minimize the contribution from tyrosine (Tyr). The fluorescence emission was collected from 300 to 600 nm. The fluorescence of serum albumins was corrected for the inner filter effect due to absorbance by BPS at the excitation and emission wavelengths, using the following equation:17
Chart 1. Structure of BPA (1) and BPS (2)
The intrinsic fluorescence of protein is from three aromatic amino acids, phenylalanine, tyrosine, and tryptophan.10 The dominant fluorophore among this is tryptophan. Normally only one or a few tryptophans is present in a protein, which makes the spectral interpretation possible. Though tyrosine has considerable quantum yield, its contribution can totally be avoided by keeping the excitation wavelength in the region 290−305 nm. Owing to its two nearby isoenergic transitions, tryptophan fluorescence is highly sensitive to local environment.10b Human serum albumin (HSA) is a single polypeptide chain having 585 amino acid residues, and has only one tryptophan.11 As spectroscopic techniques are nondestructive, the interaction study of protein−pollutant under various experimental conditions may provide valuable information on the conformational transitions. The studies on the interaction between drugs and biomacromolecules have been extensively reported,12 but very little attention has been paid to the interactions between toxic compounds and biomacromolecules. Studies on the binding of fungicide propiconazole and herbicide glyphosate to human serum albumin13 have been shown to cause some conformational and microenvironmental change in HSA. Phthalates are also known to cause protein destabilization and partial unfolding.14 The interaction of BPA at a concentration range of 5.0 × 10−5 to 5.0 × 10−4 M with HSA has been reported recently.3b This study indicated dynamic quenching for HSA−BPA interaction with the involvement of hydrophobic forces as the main type of interaction. They have also shown that the interaction of BPA stabilized the secondary structure of protein to some degree. Since most of the investigations identify the presence of BPS in articles that one comes across on a daily basis, it is worthwhile to study its possible interaction with HSA. The comparative studies with BPA−HSA interaction also might bring about new scientific insights about the close analogues. In the present work, we have investigated the effect of two structurally analogous bisphenolsBPS and BPAon serum albumins (HSA and BSA) under physiological conditions. Bovine serum albumin (BSA), that has a close similarity to HSA in sequence and conformation, is also utilized in this study.15 The difference with HSA is mainly the presence of two tryptophan residues.16 The changes brought in by these EDCs on proteins are studied using steady-state, synchronous, and time-resolved fluorescence spectroscopy (TRF) and circular dichroism (CD) spectroscopy. This study tries to unveil the mechanisms underlying the interaction, micropolarity of the environment, and fluorescence resonance energy transfer (FRET). The work has also addressed the protective effect of small amounts of anionic surfactant SDS on BPA-induced denaturation and has been manifested simply through the emission spectral and excited state lifetime behavior of serum albumins.
⎡ A + Aems ⎤ Fcorr = Fobs × antilog⎢ ex ⎥⎦ ⎣ 2
(1)
where Fcorr and Fobs are the corrected and observed fluorescence intensities, respectively, and Aex and Aems are the absorbance at excitation and emission wavelengths, respectively. For synchronous fluorescence measurements, the emission range was between 240 and 320 nm and Δλ was set at 15 and 60 nm. 2.3. Time-Resolved Fluorescence Measurements. Time-resolved fluorescence decay measurements were carried out by time-correlated single photon counting using a Horiba Jobin Yvon spectrometer. A pulsed diode (λmax = 295 nm) was used as the excitation source, and emission was monitored at respective emission wavelengths. The data was analyzed by using DAS6 software attached with the system. 2.4. Circular Dichroism Spectroscopy Measurements. Circular dichroism (CD) measurements were recorded with a Jasco J-815 spectropolarimeter. CD measurements were performed at 298 K. Spectra were collected from 200 to 250 nm with a scan speed of 200 nm/min with a spectral bandwidth of 10 nm using a 1 cm path length. Each spectrum was the average of four scans. The serum albumin concentration was kept constant (1 μM) while varying the BPS concentration. The results are expressed as MRE (mean residue ellipticity) in deg cm2 dmol−1, which is given by MRE =
observed CD (mdeg) Cpnl
(2)
where Cp is the molar concentration of the protein, n is the number of amino acid residues (585 and 582 in the case of HSA and BSA), and l is the path length (1 cm). The α-helical content of free and bound protein was eventually evaluated from the MRE value at 209 nm using the formula α‐helix % =
2. EXPERIMENTAL SECTION 2.1. Materials. Human serum albumin (HSA), bovine serum albumin (BSA), bisphenol S (BPS), and bisphenol A (BPA) were purchased from Sigma-Aldrich and were used
[−MRE 209 − 4000] [33000 − 4000]
× 100
(3)
where MRE209 is the observed MRE value at 209 nm, 4000 is the MRE of the β-form and random coil conformation at 209 nm, and 33000 is the MRE value of a pure α-form at 209 nm. 3833
dx.doi.org/10.1021/jp500404u | J. Phys. Chem. B 2014, 118, 3832−3843
The Journal of Physical Chemistry B
Article
Figure 1. Fluorescence spectra showing quenching of intrinsic fluorescence of HSA (I-A) and BSA (I-B) with an increase in BPS concentration. [HSA] = [BSA] = 10 μM; curves 1−14: [BPS] = 0, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.5, 3.5, and 4 μM; λexc = 295 nm; pH 7.4; T = 298 K. The dashed line shows the emission spectrum of BPS only (2 μM).
3. RESULTS AND DISCUSSION 3.1. Fluorescence Quenching Studies. The molecular interaction of BPS to the fluorophore groups in HSA and BSA is studied using fluorescence spectroscopy. In order to selectively excite the Trp, the excitation was carried out at 295 nm.10b,12a The steady-state fluorescence spectrum is widely applied to monitor the local microenvironment in and around the Trp amino acid residue because of its high sensitivity to the local environment.18 Therefore, any modulation in emission spectra of tryptophan of native protein can be used to examine the conformational events and ligand-binding properties of proteins. The fluorescence spectra of HSA (IA) and BSA (IB) at pH 7.4 for the successive addition of BPS are presented in Figure 1. In the absence of BPS, the emission maximum of HSA and BSA in Tris buffer was centered at 341 nm, and 345 nm identifies a signature for the Trp residue embedded in a hydrophobic microenvironment within the native protein.19 Pure BPS solution did not show any detectable fluorescence in this region. The successive addition of BPS results in the drop of fluorescence intensity of HSA at 341 nm with a concomitant formation of a new emission band centered at ∼412 nm. The intensity of the band at 412 nm increases up to a BPS concentration of 1.6 μM (indicated as an orange curve). However, further increase in the concentration of BPS (1.6−4 μM) led to a significant drop in fluorescence emission at 412 nm. These observations suggest the possibility of selfquenching. In the case of BSA, on adding BPS, a similar fluorescence quenching pattern like HSA is observed with the appearance of a broad band centered at 476 nm (Figure 1B). The intensity of the band at 476 nm increases up to a BPS concentration of 1.6 μM even though the newly formed band is less intense. Here also the intensity of the new band starts diminishing upon further addition of BPS (1.6−4 μM). In order to understand the saturating concentration range of BPS for binding with serum albumins, the fluorescence intensity of HSA at 412 nm and BSA at 476 nm was plotted against the concentration of BPS. From the plot (Figure 2), it is evident that, with a protein concentration of 10 μM, the binding of BPS to serum albumins saturates at around 1.6 μM.
Figure 2. Comparison of variation in the fluorescence intensity of HSA and BSA (at 414 and 476 nm, respectively) against the varying concentrations of BPS.
BPS interacts with serum albumins without making any change in its emission wavelength. Addition of BPS at very low concentration (micromolar range) leads to an efficient quenching of protein fluorescence. This super quenching may be due to a higher degree of interaction between the molecules. The quenching and subsequent formation of a new emission band (at 412 nm for HSA and 476 nm for BSA) indicates the possibility of energy transfer between serum albumins and BPS, or it is highly probable that the BPS forms a complex with the serum albumins which displays at higher wavelengths.20 There is a saturation limit for BPS to form a complex with both HSA and BSA. Beyond that, the added BPS can remain in the free or “unbound” form in the system. The presence of a free BPS molecule can result in the “self-quenching” which is responsible for the observed decline of the new band beyond the saturating concentration. A similar concentration dependent interaction behavior is reported in the case of tetracycline.12a Addition of BPS beyond the saturation limit can lead to a collisional encounter and energy transfer between the analytes (serum albumin and serum albumin−BPS complex). 3834
dx.doi.org/10.1021/jp500404u | J. Phys. Chem. B 2014, 118, 3832−3843
The Journal of Physical Chemistry B
Article
Figure 3. Corrected Stern−Volmer plots for (A) HSA and (B) BSA against varying concentrations of BPS at 298 K.
Table 1. Quenching Constants and Binding Parameters of Protein−BPS Systems at 298 K KSV (M−1) 5.214 × 10 6.52 × 105 5
HSA BSA
Kq (M−1 s−1)
n1
13
5.214× 10 6.52 × 1013
1.01 1.04
Kq =
KSV τ0
Kb1 (M−1)
Kb2 (M−1)
0.399 0.319
7.91 × 10 1.17 × 106
2.545 × 102 0.845 × 102
5
When small molecules bind independently to a set of equivalent sites on a macromolecule, the binding constant (Kb) and the number of binding sites (n) can be determined using the following equation
At this stage, it is essential to explain the mechanism of BPS induced quenching of intrinsic tryptophanyl fluorescence of serum albumins. The quenching mechanisms may be either dynamic (collisional) or static quenching (ground state complex formation).10b In order to understand the nature of the quenching mechanism of serum albumins in the presence of bisphenols, the Stern−Volmer (SV) equation has been used for the analysis.10b F0 = 1 + Kqτ0[Q] = 1 + KSV[Q] F
n2
⎡ (F − F ) ⎤ log⎢ 0 ⎥ = log Kb + n log[Q] ⎣ ⎦ F
(6)
where Kb reflects the degree of interaction of HSA and BSA with BPS. The plots of log[(F0 − F)/F] versus log[Q] for the HSA−BPS and BSA−BPS have two regression curves (Figure S1 of the Supporting Information). The corresponding Kb and n values are estimated and are summarized in Table 1. At lower concentrations of BPS where the binding site n1 ≈ 1, both the protein and BPS forms a 1:1 complex. Moving to higher concentrations of BPS, it is found that the value of binding site n2 is lower than 1 for both of the serum albumins. Such an “n” value (
