Chemosphere 119 (2015) 590–600

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Interaction mechanisms between organic UV filters and bovine serum albumin as determined by comprehensive spectroscopy exploration and molecular docking Junjie Ao a, Li Gao a,b, Tao Yuan a,⇑, Gaofeng Jiang c a b c

School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China School of Resource and Environment, Ningxia University, Yinchuan 750021, China School of Public Health, Medical College, Wuhan University of Science and Technology, Wuhan, Hubei 430065, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 BSA fluorescence is quenched by five

commonly-used UV filters.  UV filters can extend the BSA

Fluorescence Quenching

polypeptide chain and disrupt its ahelical stability.  UV filter molecules bind in site II (subdomain IIIA) of BSA by docking.

Molecular Docking

Interaction with serum albumin Conformation Change

a r t i c l e

i n f o

Article history: Received 23 April 2014 Received in revised form 13 June 2014 Accepted 5 July 2014

Handling Editor: Caroline Gaus Keywords: Interaction mechanisms Organic UV filters Bovine serum albumin Spectroscopy Molecular docking

a b s t r a c t Organic UV filters are a group of emerging PPCP (pharmaceuticals and personal care products) contaminants. Current information is insufficient to understand the in vivo processes and health risks of organic UV filters in humans. The interaction mechanism of UV filters with serum albumin provides critical information for the health risk assessment of these active ingredients in sunscreen products. This study investigates the interaction mechanisms of five commonly used UV filters (2-hydroxy-4-methoxybenzophenone, BP-3; 2-ethylhexyl 4-methoxycinnamate, EHMC; 4-methylbenzylidene camphor, 4-MBC; methoxydibenzoylmethane, BDM; homosalate, HMS) with bovine serum albumin (BSA) by spectroscopic measurements of fluorescence, circular dichroism (CD), competitive binding experiments and molecular docking. Our results indicated that the fluorescence of BSA was quenched by these UV filters through a static quenching mechanism. The values of the binding constant (Ka) ranged from (0.78 ± 0.02)  103 to (1.29 ± 0.01)  105 L mol1. Further exploration by synchronous fluorescence and CD showed that the conformation of BSA was demonstrably changed in the presence of these organic UV filters. It was confirmed that the UV filters can disrupt the a-helical stability of BSA. Moreover, the results of molecular docking revealed that the UV filter molecule is located in site II (sub-domain IIIA) of BSA, which was further confirmed by the results of competitive binding experiments. In addition, binding occurred mainly through hydrogen bonding and hydrophobic interaction. This study raises critical concerns regarding the transportation, distribution and toxicity effects of organic UV filters in human body. Ó 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Address: No. 800 Dongchuan Road, Minhang District, Shanghai 200240, China. Tel.: +86 21 54742823. E-mail address: [email protected] (T. Yuan). http://dx.doi.org/10.1016/j.chemosphere.2014.07.019 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.

Subdomains IIIA (site II)

J. Ao et al. / Chemosphere 119 (2015) 590–600

1. Introduction Organic UV filters are increasingly used in personal care products to absorb UV radiation and protect human skin from direct exposure to the deleterious UV wavelengths of sunlight (Giokas et al., 2007). Although they have been listed as emerging contaminants, the production volume of UV filters has dramatically increased in recent decades. For example, the production capacity of benzophenone derivatives (BPs) in China was reported to be 4000 tons per year by ChangfengChem, the world’s largest manufacturer of BPs (Wang et al., 2013). Currently, there are 28 UV filters that have been used in sunscreens in Europe (Zenker et al., 2008). In China, the Hygienic Standard for Cosmetics (2007) stipulated that these 28 UV filters can be used in cosmetics (Gao et al., 2012). UV filters are continuously discharged into the environment due to the wide use of personal care products. Since most of these UV filters are highly lipophilic and minimally degradable, they are readily accumulated in the environment (Buser et al., 2006). At present, UV filters have been identified in the inlet and outlet waters of sewage treatment plants and in lake, river and ocean water. The maximum environmental concentration of UV filters has reached the level of lg L1 and is rising. For example, Nguyen et al. (2011) reported that 2-hydroxy-4-methoxybenzophenone (BP-3) and 2-ethylhexyl 4-methoxycinnamate (EHMC) ranged from 33 to 118 ng L1 and 25 to 83 ng L1, respectively, in seawater at four beaches in Italy. Negreira et al. (2009) found that 4-methylbenzylidene camphor (4-MBC) and EHMC were ubiquitous in indoor dust in Spain, with maximum concentrations of 15 lg g1. To date, research has mainly focused on the ecotoxicity and endocrine disrupting effects of organic UV filters. For example, Sieratowicz et al. (2011) investigated the effects of BP-3, EHMC and 4-MBC on the green alga Desmodesmus subspicatus and the crustacean Daphnia magna. The results showed that exposure to the aforementioned three UV filters could result in growth inhibition of D. subspicatus. Christen et al. (2011) evaluated the potential hormonal effects of EHMC on Pimephales promelas and demonstrated that EHMC could induce multiple, though low, hormonal activities in fish. Kaiser et al. (2012) investigated the effects of butyl methoxydibenzoylmethane (BDM) and EHMC on Potamopyrgus antipodarum and Melanoides tuberculata. The results showed that EHMC caused a toxic effect on reproduction in both types of snails, with lowest observed effect concentrations (LOEC) of 0.4 mg kg1 (P. antipodarum) and 10 mg kg1 (M. tuberculata). In addition, our previous study found that BP-3 and 4-MBC could inhibit the growth of Tetrahymena thermophila and could lead to a significant increase in catalase (CAT) activities in the T. thermophila cells at environmentally relevant concentrations of 1.0 lg L1 (Gao et al., 2013). UV filters have low acute toxicity in animals, but less is known about their chronic toxicity to humans from topical application. Treffel and Gabard (1996) measured the skin penetration profiles of BP-3 through tape stripping experiments. The results suggested that BP-3 penetrated into the human epidermis. Kasichayanula et al. (2007) found that BP-3 could reach blood circulation and be excreted in urine. Similarly, Soeborg et al. (2007) used a porcine skin model to assess the potential human health risk of 4-MBC and 3-BC. The data showed that the skin permeation could cause these two UV filters to reach blood vessels in the deeper skin cell layers. Some commonly-used UV filters such as BP-3, 4-MBC, EHMC, BDM and homosalate (HMS) have been identified in human urine, feces, blood, milk and placenta (Chisvert et al., 2012). Furthermore, the occurrence of UV filters in indoor dust confirmed indirect exposure to these compounds through respiratory or ingestion pathways (Negreira et al., 2009; Wang et al., 2013). Thus, it is imperative to

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reassess the potential health risks of UV filters. Unfortunately, there is not sufficient information about the in vivo behaviors of UV filters, such as their interaction and binding affinity towards albumin in blood. The investigation of these behaviors is important to understanding the distribution, metabolism and toxicity of UV filters in human beings. In recent years, studies have been carried out on the interactions between environmental contaminants and carrier proteins (D’Alessandro et al., 2013; Ng and Hungerbuhler, 2013; Zhang et al., 2013). As the most abundant carrier protein in plasma, serum albumin accounts for about 60% of the total protein in plasma. Serum albumin can combine with many endogenous or exogenous compounds and plays a role in storage and transport. Due to its higher homology with human serum albumin (HSA) and relatively inexpensive cost, bovine serum albumin (BSA) has the advantage of allowing more thorough experimental measurements and investigations than carrier proteins, especially for emerging contaminants, such as perfluorinated chemicals and pharmaceuticals (Qin et al., 2010; D’Alessandro et al., 2013). However, as mentioned above, information is scarce regarding the interaction between UV filters and serum albumin, which is a critical factor in understanding the transport, distribution and toxicity of UV filters in the human body. Only recently, Zhang et al. (2013) investigated the potentially toxic interactions of several benzophenones with HSA. To our knowledge, there is no report on serum albumin interaction with commonly-used UV filters such as 4-MBC, BDM, EHMC and HMS. This study attempts to investigate the interactions between these UV filters and serum albumin. Spectroscopic methods and molecular docking are valuable tools for determining the interactions between small molecules and bio-macromolecules. They allow non-intrusive measurement of substances at low concentrations under physiological conditions (Gong et al., 2012). In this study, we investigated the interactions of five commonly used organic UV filters, BP-3, 4-MBC, BDM, EHMC and HMS, with BSA by multiple spectroscopic techniques and molecular docking. This study provides basic data for clarifying the binding mechanisms of organic UV filters with BSA. It will also help to improve understanding of the mode of toxicity and reduce the shortage of information on health risk assessment for these active ingredients in sunscreen products.

2. Materials and methods 2.1. Chemicals BSA (98% purity) and BP-3 (98% purity) were purchased from Sigma–Aldrich (St. Louis, MO, USA). 4-MBC (99% purity) was purchased from Alfa Aesar (USA). BDM (95% purity), EHMC (96% purity), HMS (98% purity), warfarin (98% purity) and ibuprofen (98% purity) were purchased from TCI (Tokyo, Japan). The structures and relevant physio-chemical properties of the UV filters are given in Table 1. BSA stock solution (1.0  105 M) was prepared in 0.01 M phosphate buffer (pH = 7.40) for circular dichroism (CD) measurements and in 0.10 M Tris–HCl buffer (pH = 7.40, 0.1 M NaCl) for the remaining experiments. Each stock solution of BP-3, 4-MBC, BDM, EHMC, HMS, warfarin and ibuprofen (0.01 M) was prepared in DMSO. The stock solutions were kept in the dark at 4 °C. All other reagents were of analytical grade unless otherwise stated. Ultrapure water (18.2 MX, Rephile, Shanghai, China) was used throughout all experiments. All measurements of pH were conducted using a PHS-3C acidity meter (Shanghai Zhiguang Instruments & Measuring Appliance Co., Ltd., China).

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Table 1 Structures and some physic-chemical properties of the tested UV filters. Molecular weight

CAS

log Kowa

Solubility (mg L1)

BP-3

228.25

131-57-7

3.79

68.56

4-MBC

254.38

36 861-47-9

5.92

0.20

BDM

310.40

70 305-09-1

4.51

1.52

EHMC

290.41

5466-77-3

5.80

0.15

HMS

262.35

118-56-9

6.16

0.42

Compounds

a

Chemical structure

log Kow, the logarithmised octanol–water partition coefficient, calculated from EPISUITE 4.11. (http://www.epa.gov/oppt/exposure/pubs/episuitedl.htm).

2.2. Fluorescence quenching First, 0.5 mL BSA, 2.0 mL Tris–HCl buffer and differing concentrations of UV filter solutions were added to a 10 mL volumetric flask. The mixtures were then diluted to 10 mL with ultrapure water to give final UV filter concentrations of 0.0, 0.5, 1.0, 2.0, 4.0, 6.0 and 8.0  106 M. A HWS12 electric-heated thermostatic water bath (Shanghai Yiheng Scientific Instruments Co., Ltd., China) was used to control the temperature of the mixtures at 22, 27, 32, 37 and 42 °C. Fluorescence spectra and intensities were recorded on a RF-5301PC Spectrofluorimeter (Shimadzu, Japan) with a 1 cm quartz cell. Both the excitation and emission slit widths were set at 5 nm. The emission spectra were recorded in the range of 300–500 nm with an excitation wavelength of 280 nm. To eliminate the possibility of inter-filter effects of proteins and ligands, the absorbance measurements were performed to correct the measured F values using the equation (Zhang et al., 2010):

F e ¼ F m eðA1 þA2 Þ=2

ð1Þ

where Fe and Fm are the corrected and measured fluorescence, respectively, and A1 and A2 are the sum of the absorbance of proteins and ligands at excitation and emission wavelengths, respectively. 2.3. Competitive binding experiments The experiments were performed using warfarin and ibuprofen as site markers. Warfarin and ibuprofen mark site I and site II, respectively. 0.5 mL BSA, 0.5 mL warfarin or ibuprofen, 2.0 mL Tris–HCl buffer and differing concentrations of UV filter solutions were added to 10 mL volumetric flasks. The mixtures were then diluted to 10 mL with ultrapure water to give final UV filter concentrations of 0.0, 0.5, 1.0, 2.0, 4.0, 6.0 and 8.0  106 M. The fluorescence emission spectra were recorded at 27 °C. 2.4. Synchronous fluorescence Synchronous fluorescence measurements were performed on a RF-5301PC Spectrofluorimeter (Shimadzu, Japan) with a 1 cm quartz cell. Synchronous fluorescence spectra for BSA with various

concentrations of UV filters were obtained in the ranges of 235– 400 nm (Dk = 15 nm) and 280–400 nm (Dk = 60 nm) with excitation and emission slit widths of 5 nm. 2.5. Circular dichroism (CD) CD measurements were made on a J-815 Circular Dichroism Spectrometer (Jasco, Tokyo, Japan) at room temperature using a 0.1 cm quartz cell and with a scanning speed of 50 nm min1. The spectra were recorded over a wavelength range of 190– 250 nm, and each spectrum represents the average of three successive scans. The BSA concentration was 1.0  106 M while concentrations of UV filters were 0.0, 2.0 and 4.0  106 M. The phosphate buffer solution was used as a blank and automatically subtracted from the sample scans. 2.6. Molecular docking The crystal structure of BSA (P02769) was obtained from the SWISS-MODEL Repository (http://swissmodel.exp-asy.org/repository/?pid=smr01&zid=async) (Shi et al., 2013). The structure of the ligand was obtained from PubMed NCBI website (http://pubchem.ncbi.nlm.nih.gov/). AutoDock 4.2 (http://autodock.scripps.edu/, free software) was used to calculate the interaction modes between UV filters and BSA. All calculations were carried out on a 3.3 GHz Intel Core i3based machine running MS Windows XP SP3 as operating system. The docking results were illustrated by PyMol (http://www.pymol.org/) and Discovery Studio Visualizer 2.5 (Accelrys, free version). 3. Results and discussion 3.1. Fluorescence quenching mechanism The fluorescence of BSA comes from tryptophan, tyrosine and phenylalanine residues. The characteristics of BSA intrinsic fluorescence are very sensitive to its microenvironment, and a variety of molecular interactions can diminish the fluorescence intensity of proteins, which is called fluorescence quenching (Lakowicz, 2006). Fluorescence quenching of the proteins can be used to

J. Ao et al. / Chemosphere 119 (2015) 590–600

retrieve information about the changes in the molecular microenvironment in the vicinity of the chromophore molecules. Fig. 1 shows the fluorescence emission spectra of BSA in the absence and presence of various concentrations of UV filters at 27 °C. It was noted that, when fixing the excitation wavelength at 280 nm, BSA had a strong fluorescence emission band at 340 nm. In addition, with increasing concentrations of UV filters, the fluorescence intensities of BSA decreased uniformly, but the shape of the spectra did not change. This result indicated that a UV filter–BSA complex had formed to quench the fluorescence of BSA. Normally, quenching occurs by different mechanisms, usually classified as static quenching, dynamic quenching or non-radiative energy transfer (Zhu et al., 2012). Dynamic quenching is collisional

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quenching, which is enhanced by increasing temperature and diminished by increasing viscosity. Static quenching refers to the interaction of a fluorophore with a quencher to form a stable non-fluorescent complex (Gong et al., 2012). To better understand the fluorescence quenching mechanism of the selected UV filters in this study, further investigations were conducted. For dynamic quenching, the mechanism can be described by the Stern–Volmer equation:

F 0 =F ¼ 1 þ K q s0 ½Q  ¼ 1 þ K sv ½Q 

ð2Þ

where F0 and F represent the fluorescence intensities in the absence and presence, respectively, of the quencher; Kq is the biomolecular quenching rate constant; Ksv is the Stern–Volmer quenching constant; [Q] is the concentration of the quencher; and s0 is the average

Fig. 1. Fluorescence spectra of UV filters-BSA system at 27 °C, pH 7.40. From 1 to 7: The concentration of BSA was 5.0  107 M, UV filters concentration corresponding to 0.0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0  106 M, respectively.

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fluorescence lifetime of the biomolecule without the quencher. The fluorescence lifetime of the biopolymer was reported as 108 s (Lakowicz and Weber, 1973). Eq. (2) was applied to determine Ksv by a linear regression plot of F0/F vs. [Q]. The Stern–Volmer plots for the quenching of BSA by UV filters at five temperatures (22, 27, 32, 37 and 42 °C) are shown in Fig. S1, and the values of Kq and Ksv are listed in Table 2. It was noted that the Stern–Volmer quenching constant Ksv is inversely correlated with temperature. The results indicated that the quenching was probably not initiated by dynamic collision but from compound formation. Moreover, the value of Kq gave further information about the mechanism of quenching. It was reported that the maximum scatter collision quenching constant Kq of various quenchers with a biopolymer is 2.0  1010 L mol1 s1 (WR, 1962). However, in the present study, the values of Kq were higher by nearly three orders of magnitude (Table 2). The Kq value results further suggested that the probable fluorescence quenching mechanism of BSA by UV filters was static quenching. 3.2. Binding constants and binding sites For static quenching, when small molecules bind independently to a set of equivalent sites on a macromolecule, the apparent binding constant Ka and binding site n can be described by the equation (Abert et al., 1993):

lg½ðF 0  FÞ=F ¼ lgK a þ nlg½Q 

ð3Þ

where Ka and n are the apparent binding constant and the number of binding sites, respectively, and F0, F and [Q] have the same meanings as in Eq. (2). The corresponding double logarithm curves of lg[(F0  F)/F] versus lg[Q] for the UV filter–BSA systems are shown in Fig. S2. The relevant calculated values are listed in Table 3. For the binding parameters, it was observed that the values of n were approximately equal to 1, indicating the existence of a single binding site for UV filters on BSA (Table 3). The estimated values of Ka were from (0.78 ± 0.02)  103 to (1.29 ± 0.01)  105 L mol1, indicating that there was a moderately strong attraction between

the UV filters and BSA. In addition, BDM might have a higher binding affinity with BSA and form a much more stable non-fluorescent complex with BSA, as its Ka value is higher than those of the other UV filters (Table 3). Based on the values of Ka, the binding affinities of UV filters were in the order of BDM > BP-3 > 4MBC > EHMC > HMS. The binding constants obtained with Eq. (3) were applied to the following calculations of binding modes. 3.3. Thermodynamic parameters and binding modes Because the binding constant is dependent on temperature, a thermodynamic process was assumed to be responsible for the formation of the complex. Therefore, the thermodynamic parameters dependent on temperature were analyzed in order to characterize the forces acting between the UV filters and BSA. Essentially, there are four main types of non-covalent interactions in the binding of a ligand to proteins, including hydrogen bonds, van der Waals force, electrostatic force, and hydrophobic interaction (He and Carter, 1992). Thermodynamic parameters, such as enthalpy (DH), entropy (DS) and free energy change (DG), are the main variables used to estimate the binding mode and confirm the interaction forces. The thermodynamic parameters are evaluated using the van’t Hoff equation:

lnK ¼ DH0 =RT þ DS0 =R

ð4Þ

where K is equivalent to the binding constant Ka obtained from Eq. (3) and R is the gas constant, and the free energy change (DG0) is determined from the relationship:

DG0 ¼ DH0  DTS0

ð5Þ 0

The negative value for free energy change (DG ) supported the assertion that the binding process is spontaneous and that a high temperature promotes a spontaneous interaction (Samari et al., 2012). According to Ross (Ross and Subramanian, 1981), the signs and magnitudes of the thermodynamic parameters (DH0 and DS0) for protein reactions can account for the main forces contributing

Table 2 Stern–Volmer quenching constant (Ksv) and biomolecular quenching rate constant (Kq) of UV filters-BSA system at different temperatures, pH 7.40. Quencher

a

T (°C)

Ksv (L mol1)

Ra

Kq (L mol1 s1) 5

13

BP-3

22 27 32 37 42

(1.11 ± 0.02)  10 (1.03 ± 0.02)  105 (0.86 ± 0.01)  105 (0.78 ± 0.01)  105 (0.68 ± 0.02)  105

(1.11 ± 0.02)  10 (1.03 ± 0.02)  1013 (0.86 ± 0.01)  1013 (0.78 ± 0.01)  1013 (0.68 ± 0.02)  1013

0.999 0.999 0.998 0.999 0.999

4-MBC

22 27 32 37 42

(1.31 ± 0.03)  105 (0.99 ± 0.02)  105 (0.72 ± 0.01)  105 (0.51 ± 0.01)  105 (0.33 ± 0.01)  105

(1.31 ± 0.03)  1013 (0.99 ± 0.02)  1013 (0.72 ± 0.01)  1013 (0.51 ± 0.01)  1013 (0.33 ± 0.01)  1013

0.996 0.997 0.997 0.998 0.997

BDM

22 27 32 37 42

(1.42 ± 0.02)  105 (1.16 ± 0.01)  105 (1.02 ± 0.01)  105 (0.87 ± 0.03)  105 (0.72 ± 0.02)  105

(1.42 ± 0.02)  1013 (1.16 ± 0.01)  1013 (1.02 ± 0.01)  1013 (0.87 ± 0.03)  1013 (0.72 ± 0.02)  1013

0.993 0.995 0.999 0.999 0.997

EHMC

22 27 32 37 42

(1.50 ± 0.02)  105 (1.33 ± 0.01)  105 (1.07 ± 0.01)  105 (0.92 ± 0.01)  105 (0.78 ± 0.01)  105

(1.50 ± 0.02)  1013 (1.33 ± 0.01)  1013 (1.07 ± 0.01)  1013 (0.92 ± 0.01)  1013 (0.78 ± 0.01)  1013

0.992 0.993 0.998 0.997 0.994

HMS

22 27 32 37 42

(3.61 ± 0.05)  104 (2.98 ± 0.03)  104 (2.21 ± 0.03)  104 (1.62 ± 0.01)  104 (1.32 ± 0.02)  104

(3.61 ± 0.05)  1012 (2.98 ± 0.03)  1012 (2.21 ± 0.03)  1012 (1.62 ± 0.01)  1012 (1.32 ± 0.02)  1012

0.997 0.999 0.997 0.999 0.998

R is the linear correlated coefficient of the linear regression plots.

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J. Ao et al. / Chemosphere 119 (2015) 590–600 Table 3 Binding and thermodynamic parameters of UV filters-BSA system at different temperatures, pH 7.40. Quencher BP3

4MBC

BDM

EHMC

HMS

a

T (°C)

Ka (L mol1) 4

n

Ra

22 27 32 37 42

(5.52 ± 0.08)  10 (4.14 ± 0.07)  104 (2.33 ± 0.07)  104 (1.46 ± 0.05)  104 (1.02 ± 0.02)  104

0.94 ± 0.01 0.93 ± 0.01 0.90 ± 0.01 0.88 ± 0.01 0.86 ± 0.01

0.999 0.998 0.995 0.999 0.999

22 27 32 37 42

(3.03 ± 0.05)  104 (2.51 ± 0.03)  104 (1.82 ± 0.03)  104 (1.41 ± 0.03)  104 (1.18 ± 0.02)  104

0.87 ± 0.01 0.88 ± 0.01 0.89 ± 0.01 0.89 ± 0.02 0.91 ± 0.01

0.994 0.998 0.997 0.998 0.995

22 27 32 37 42

(1.29 ± 0.01)  105 (1.63 ± 0.02)  105 (1.97 ± 0.01)  105 (2.60 ± 0.01)  105 (3.08 ± 0.02)  105

0.99 ± 0.01 1.02 ± 0.01 1.05 ± 0.01 1.10 ± 0.02 1.13 ± 0.01

0.997 0.996 0.999 0.996 0.999

22 27 32 37 42

(1.15 ± 0.03)  104 (0.85 ± 0.01)  104 (0.77 ± 0.02)  104 (0.63 ± 0.01)  104 (0.49 ± 0.01)  104

0.77 ± 0.01 0.76 ± 0.01 0.76 ± 0.01 0.75 ± 0.01 0.74 ± 0.01

0.999 0.999 0.999 0.993 0.993

22 27 32 37 42

(1.95 ± 0.05)  103 (1.68 ± 0.03)  103 (1.33 ± 0.02)  103 (1.01 ± 0.02)  103 (0.78 ± 0.02)  103

0.74 ± 0.01 0.75 ± 0.01 0.75 ± 0.02 0.76 ± 0.01 0.78 ± 0.02

0.995 0.999 0.997 0.997 0.995

DH0 (kJ mol1)

68.38 ± 0.12

38.01 ± 0.09

34.05 ± 0.05

30.86 ± 0.09

35.98 ± 0.11

DS0 (J mol1 K1)

DG0 (kJ mol1)

140.48 ± 0.56

26.94 ± 0.42 26.24 ± 0.42 25.54 ± 0.42 24.83 ± 0.42 24.13 ± 0.42

42.89 ± 0.37

25.35 ± 0.31 25.14 ± 0.31 24.92 ± 0.31 24.71 ± 0.31 24.50 ± 0.31

213.21 ± 0.66

28.85 ± 0.18 29.92 ± 0.18 30.98 ± 0.18 32.05 ± 0.18 33.11 ± 0.18

27.06 ± 0.29

22.88 ± 0.33 22.75 ± 0.33 22.61 ± 0.33 22.48 ± 0.33 22.34 ± 0.33

58.52 ± 0.53

18.72 ± 0.35 18.43 ± 0.35 18.13 ± 0.35 17.84 ± 0.35 17.55 ± 0.35

R is the linear correlated coefficient of the linear regression plots.

to protein stability. From a thermodynamic standpoint, DH0 > 0 and DS0 > 0 implies hydrophobic interaction, DH0 < 0 and DS0 < 0 reflects the van der Waals force or hydrogen bond formation and DH0 < 0 and DS0 > 0 suggests an electrostatic force. The values of DH0 and DS0 were obtained from the linear relationship between lnK and the reciprocal of absolute temperature (1/T). The thermodynamic parameters are presented in Table 3. The negative values for free energy (DG0) indicated that the binding process was spontaneous. For BP-3, 4-MBC, EHMC and HMS, the negative DH0 and DS0 values indicated that the binding process investigated here was driven mainly by the van der Waals force and hydrogen bonding. However, for BDM, both the DH0 and DS0 values were positive; therefore, the hydrophobic force played a major role in its binding process. 3.4. Conformational investigations 3.4.1. Synchronous fluorescence spectroscopy Despite ascertaining that the binding of UV filters to BSA caused the fluorescence quenching of BSA, it was still unknown whether this binding affected the conformation or molecular environment of BSA. In this study, therefore, synchronous fluorescence was utilized to further verify the interaction of UV filters with BSA. Synchronous fluorescence spectroscopy is a sensitive technique used to analyze the microenvironmental changes of chromophores (Yue et al., 2008). The synchronous fluorescence spectra present information about the molecular microenvironment in the vicinity of fluorophore functional groups (Lu et al., 2010). When Dk was established at 60 and 15 nm, the synchronous fluorescence experiments gave data which were characteristic of tryptophan and tyrosine residues, respectively (Teng et al., 2011). As shown in Fig. S3, the maximum fluorescence intensities were from 300 to 500 when Dk = 60 nm but only from 50 to 150 when Dk = 15 nm. For all of the tested UV filters, the quenching of the fluorescence intensity of tryptophan residue was stronger than that of tyrosine residue, suggesting that tryptophan residues contribute greatly to the

quenching of the intrinsic fluorescence of BSA. In addition, the shift in the position of the maximum emission wavelength corresponded to the changes in polarity around the chromophore molecules (Wang et al., 2007). However, no obvious wavelength shift for tyrosine or tryptophan residues was observed, as shown in Fig. S3. The results indicated that the microenvironment of the tyrosine and tryptophan residues had no obvious changes. Moreover, it was observed that the interactions of the UV filters with BSA did not cause any microenvironmental transformation around tyrosine and tryptophan BSA residues. 3.4.2. Circular dichroism spectroscopy CD spectroscopy is a sensitive and quantitative technique to monitor the conformational changes of proteins upon interaction with a ligand (Li et al., 2013). To further understand the interactions between BSA and UV filters, CD spectra were collected from free BSA and UV filter-BSA complexes. The CD spectra of BSA exhibited two negative bands in the ultraviolet region at 208 and 222 nm, which are characteristic of an a-helix of proteins (Yang et al., 1986). The explanation is that the negative peaks between 208–209 nm and 222–223 nm both contribute to an n ? p⁄ transfer for the peptide bond of the a-helix. As shown in Fig. 2, the CD spectra of BSA in the absence and presence of UV filters were similar in shape, indicating that the structure of BSA was predominantly a-helical. Yu et al. (2014) analyzed the influence of phacolysin (PCL) on the secondary structure of BSA through CD spectroscopy. The results indicated that the amount of a-helices (%) decreased from 56.5% (free BSA) to 54.9% (PCL:BSA = 1:1), which suggested that PCL changed the secondary structure of BSA. In this paper, the secondary structure of BSA was calculated with SELCON3, provided by the DICHROWEB website (http://dichroweb.cryst.bbk.ac.uk/html). The calculated results are listed in Table 4. It was noted that the free BSA had an a-helix content of 57.2%. When the molecular ratio of BSA to UV filters was 1:2, the a-helix content was reduced to 56.6%, 56.3% and 56.7% by BP-3, 4-MBC and HMS, respectively. In addition, when the molecular ratio was 1:4, the corresponding

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Fig. 2. CD spectra of BSA in the absence and presence of UV filters at room temperature, pH 7.40 (The embedded pictures are zoomed in for the two negative bands). The concentration of BSA was 1.0  106 M, UV filters concentration corresponding to 0.0, 2.0, 4.0  106 M, respectively.

a-helix content was further decreased to 56.1%, 55.7% and 56.4%, respectively. However, for BDM and EHMC, the a-helix content was increased to 57.7% and 58.0%, respectively, when the molecular ratio of BSA to UV filters was 1:2. Moreover, the a-helix content increased to 58.2% and 58.7%, respectively, when the molecular ratio was 1:4. The decrease in percentage of a-helix structure indicated that the binding of UV filters with BSA induced a slight unfolding of the constitutive polypeptides of the protein and exposed some hydrophobic regions that were previously buried (Xu et al., 2014). The reverse occurred when the percentage increased. Thus, the binding of BSA with BP-3, 4-MBC and HMS caused BSA conformational changes with a loss in a-helical

Table 4 The secondary structure data of BSA in the absence and presence of UV filters. Molecular ratio

a-helix (%)

b-sheet (%)

b-turn (%)

Random (%)

Free BSA BSA:BP-3 = 1:2 BSA:BP-3 = 1:4 BSA:4-MBC = 1:2 BSA:4-MBC = 1:4 BSA:BDM = 1:2 BSA:BDM = 1:4 BSA:EHMC = 1:2 BSA:EHMC = 1:4 BSA:HMS = 1:2 BSA:HMS = 1:4

57.2 56.6 56.1 56.3 55.7 57.7 58.2 58.0 58.7 56.7 56.4

7.0 7.1 7.1 7.2 7.4 6.7 6.5 6.4 6.3 7.0 7.4

12.8 13.1 12.0 11.9 12.3 12.7 11.6 12.2 12.7 12.7 13.8

24.5 24.8 24.3 24.5 24.6 24.5 24.0 24.1 24.5 24.4 24.5

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stability, while BDM and EHMC caused a gain in a-helical stability. By combining the results of Fig. 2 and Table 4, it was confirmed that the binding of these UV filters to BSA caused the changes to the secondary structure of the BSA protein. Similar results were observed by Zhang et al. (2013), who observed that the binding interactions of four benzophenones with HSA might cause structural damage to HSA. 3.5. Molecular docking study Mature BSA is composed of 583 amino acids, and its three dimensional structure is similar to a heart shape. Additionally,

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the globular protein BSA consists of three structurally similar domains, I (His 27-Gln219), II (Arg220-Ile411) and III (Lys412Ala607), with each one containing two sub-domains, A and B (Paul et al., 2010; Gong et al., 2012). The principal regions of ligand binding sites in albumin are located in two hydrophobic pockets in sub-domains IIA and IIIA, namely site I and site II (Chung et al., 2007). In order to further study the binding mode and binding location of UV filters on BSA, the molecular docking of UV filters with BSA was carried out using AutoDock 4.2. The binding behaviors between the five UV filters and BSA are shown in Fig. 3. Normally, the binding site could be determined by the free energy calculated by the docking software. The lower

Fig. 3. (A) Binding site of UV filters to BSA obtained from molecular docking simulation. The BSA and UV filters were represented by ribbon structure and space-filling model, respectively. (B) Binding mode between UV filters and BSA. The amino acid residues of BSA and UV filters were represented by a stick-ball model of grey and purple, respectively. (C) Detailed illustration of the binding mode. The residues and UV filters were represented by different tinctorial stick model. The hydrogen bonds were represented using green dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3 (continued)

free energy indicates higher binding possibilities (Table S1). It was noted that the UV filter molecule was located within the binding pocket of site II (IIIA) in Fig. 3A, which demonstrated that the complex formed by the binding of UV filters to sub-domain IIIA is more stable than sub-domain IIA in terms of free energy. Moreover, competitive binding experiments were performed to identify the binding site of the UV filters in BSA. Warfarin and ibuprofen were chosen as site markers for site I and site II, respectively (Hu et al., 2014). As shown in Table S2, the calculated binding constant of the warfarin-UV filters-BSA system is (0.18–15.01)  104 L mol1, while the ibuprofen-UV filters-BSA system constant is (0.46–15.62)  102 L mol1 at 27 °C. The binding constant of the ibuprofen-UV filters-BSA system decreased sharply from (0.17– 16.3)  104 to (0.46–15.62)  102 L mol1, whereas the addition of warfarin influenced the binding constant to a lesser extent.

The results suggested that the five UV filters shared the same binding site as ibuprofen, located within the binding pocket of site II (IIIA), and further confirmed the molecular docking results. As shown in Fig. 3B, there were two hydrogen bonds between BP-3 and Leu453, Tyr434; EHMC and Arg433, Lys437; HMS and Ser512, Arg433; and one hydrogen bond between 4-MBC and Asn414; BDM and Lys437 residues of BSA, with a distance in the range of 1.9–2.2 Å. The results indicated that the formation of hydrogen bonds could decrease the hydrophilicity and increase the hydrophobicity to stabilize the UV filters–BSA system. In addition, it is important to note that the five UV filters were surrounded mainly by hydrophobic residues, such as Leu430, Leu476, Leu453, Phe511, Phe426, Gly457 and Val456 (Fig. 3C). This phenomenon implies the existence of hydrophobic interaction between UV filters and BSA binding sites. It can be concluded that the interactions

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between BP-3, 4-MBC, BDM, EHMC, HMS and BSA are primarily through hydrogen bonding and hydrophobic interaction, in agreement with the binding mode proposed in the above thermodynamic analysis. Furthermore, it was noted that the calculated binding free energy (DG0) ranged from 9.95 to 6.60 kJ mol1 (Table S1), which varied from the experimental data (29.92 ± 0.18 to 18.43 ± 0.35 kJ mol1 at 27 °C, as shown in Table 3). A possible explanation may be that the crystal structure of BSA differs from that of the solution used in this study (Gong et al., 2012).

4. Conclusions In this study, the interactions of five organic UV filters with BSA were investigated using fluorescence spectroscopy, circular dichroism spectroscopy (CD), competitive binding experiments and molecular docking. From the fluorescence measurements, it was found that the fluorescence of BSA was quenched by the UV filters through a static quenching mechanism. The binding affinities of the five UV filters were in the order BDM > BP-3 > 4MBC > EHMC > HMS, and each UV filter can form one binding site with BSA. The results of thermodynamic parameter calculations indicated that the reaction type between BP-3, 4-MBC, EHMC, HMS and BSA is primarily hydrogen bonding and the van der Waals force but is mainly hydrophobic force between BDM and BSA. In addition, the conformation of BSA was changed in the presence of these organic UV filters, as demonstrated by synchronous fluorescence and CD measurements. Furthermore, the binding site results for UV filters with BSA were successfully generated by molecular docking. The results showed that the UV filter molecules were located in site II (IIIA), confirmed by the results of competitive binding experiments. Hydrogen bonding and hydrophobic interaction contributed greatly to the interactions. In this study, both experimental results and theoretical data consistently confirmed that each selected UV filter had an effect on BSA conformation. To our knowledge, this is the first study to provide insight into the interactions of these UV filters with BSA by combining experimental and theoretical investigations. This study raises critical concerns regarding the transport, distribution and toxicity effects of organic UV filters in the human body, especially for sensitive groups, such as infants and pregnant women. Acknowledgments We acknowledge financial support from the National Science Foundation of China (No. 21277092), the National Basic Research Program of China (973 Program, No. 2014CB943300) and the Key Innovation Research Project of Shanghai Education Committee (No. 12ZZ027).

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