Binding properties and structure–affinity relationships of food antioxidant butylated hydroxyanisole and its metabolites with lysozyme

Accepted Manuscript Binding properties and structure-affinity relationships of food antioxidant butylated hydroxyanisole and its metabolites with lysozyme Di Wu, Jin Yan, Peixiao Tang, Shanshan Li, Kailin Xu, Hui Li PII: DOI: Reference:

S0308-8146(15)00734-7 http://dx.doi.org/10.1016/j.foodchem.2015.05.013 FOCH 17559

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

7 January 2015 8 April 2015 5 May 2015

Please cite this article as: Wu, D., Yan, J., Tang, P., Li, S., Xu, K., Li, H., Binding properties and structure-affinity relationships of food antioxidant butylated hydroxyanisole and its metabolites with lysozyme, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.05.013

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Binding properties and structure-affinity relationships of food antioxidant butylated hydroxyanisole and its metabolites with lysozyme Di Wu, Jin Yan, Peixiao Tang, Shanshan Li, Kailin Xu and Hui Li* College of Chemical Engineering, Sichuan University, Chengdu 610065, P. R. China Running Title: Binding properties of BHA and its metabolites with lysozyme Abstract Considering the harmful impact of food antioxidants on human bodies, thoroughly exposing their potential effects at the molecular level is important. In this study, the binding interactions of butylated hydroxyanisole (BHA), a phenolic antioxidant, and its

different

major

metabolites

tert-butylhydroquinone

(TBHQ)

and

tert-butylbenzoquinone (TBQ) with lysozyme were examined via fluorescence, three-dimensional fluorescence, circular dichroism (CD), and ligand-protein docking studies. The three compounds caused strong quenching of lysozyme fluorescence by a static quenching mechanism but with different quenching efficiencies and different effects on the α-helix content of the lysozyme. The order of binding affinity of lysozyme for all test compounds is as follows: BHA > TBQ > TBHQ. Thermodynamic parameters indicated that hydrogen bonding and van der Waals forces perform dominant functions in the binding between these compounds and lysozyme.

Furthermore,

structure–affinity relationships

*

between

the

model

Corresponding author. Address: College of Chemical Engineering, Sichuan University, Chengdu 610065, P. R. China. Tel.: +86 028 85405149; Fax: +86 028 85401207. E-mail address: [email protected](Hui, Li) 1

compounds and lysozyme were established on the basis of computational analyses. Keywords: Binding mode, Butylated hydroxyanisole, Tert-butylhydroquinone, Tert-butylbenzoquinone, Lysozyme, Molecular modeling 1. Introduction Antioxidants are used worldwide as food additives to protect foodstuffs against deterioration caused by oxidation, such as fat rancidity and colour changes (Shahidi, Janitha, & Wanasundara, 1992). Butylated hydroxyanisole (BHA) is a phenolic antioxidant that is widely used as a synthetic food additive to preserve oils and fats. BHA is also used to stabilize the freshness, nutritive value, flavour, and colour of food and animal feed products (Abiko, Miura, Phuc, Shinkai, & Kumagai, 2011). The major metabolism of BHA undergoes O-dealkylation by cytochrome P450 isozymes to produce tert-butylhydroquinone (TBHQ), another highly effective approved food antioxidant, and this hydroquinone metabolite can be further auto-oxidized to the ultimate metabolite, tert-butylbenzoquinone (TBQ), via the semiquinone radical as an intermediate (Abiko & Kumagai, 2013; Nagai, Okubo, Ushiyama, Satoh, & Kano, 1996). Historically, BHA and its metabolites have also attracted the interest of biochemists and health professionals because these compounds may aid the body in protecting itself against damage (Keum, Han, Liew, Kim, Xu, Yuan, et al., 2006; Shahidi, 1997). Notably, the use of BHA and its metabolites is not restricted to foodstuffs. Therefore, antioxidants have attracted considerable attention from multiple areas of food chemistry or toxicology. In addition, growing concern has been expressed over the potentially mutagenic and carcinogenic effects of these compounds 2

in metabolic processes from available literature (Branen, 1975; Dohrmann & Bergmann, 1995; Rodil, Quintana, & Cela, 2012; Shahabadi, Maghsudi, Kiani, & Pourfoulad, 2011; Williams, Iatropoulos, & Whysner, 1999). According to the commonly approved concept at present, the interaction of toxic compounds with physiologically important proteins contributes to understanding the effect on their absorption, transportation, distribution, and physiological function. Lysozyme, an antimicrobial proteinase with a comparatively small molecular weight (14600), is pervasive in various body fluids, such as tears, saliva, mucus, urine, lymphatic tissues, human milk and in cells of the innate immune system (Alhazmi, Stevenson, Amartey, & Qin, 2014; Mason & Taylor, 1975). According to X-ray diffraction measurements (Blake, Koenig, Mair, North, Phillips, & Sarma, 1965), lysozyme is a single, nonglycosylated polypeptide chain of 129 amino acid residues folded into two domains including six tryptophan (Trp) and three tyrosine (Tyr) residues. This protein with high natural abundance is an enzyme known for its unique ability to damage bacterial cell walls, thereby providing protection against bacterial infections (Jash & Kumar, 2014). When the diverse endogenous and exogenous ligands enter into the human body, ligand-lysozyme conjugation can be observed (Morrissey, Stocker, Wittwer, Xu, & Giacomini, 2013; Peng, Ding, Peng, Jiang, & Zhang, 2013). Therefore, lysozyme is selected to investigate the binding characteristics of BHA or its metabolites (TBHQ and TBQ), which is critical in order to understand their possible delivery, consequent availability, and relevant health risks. 3

In this work, attempts were conducted to assess the binding mechanism, binding constants, thermodynamic functions, conformational changes, and spatial structures of the complexes formed between the model compounds (BHA, TBHQ, and TBQ) and lysozyme by employing spectroscopic techniques and computational docking. 2. Materials and methods 2.1. Reagents and chemicals Lysozyme (from chicken egg white) was purchased from Sigma–Aldrich (Milwaukee, USA) and used without further purification. The lysozyme stock solution was prepared at a concentration of 1.0×10–4 mol·l–1 in 0.05 mol·l–1 Tris-HCl buffer (pH 6.5) containing 0.1 mol·l–1 NaCl, and the concentration was determined by the molar extinction coefficient value of 37750 mol–1cm–1 at 280 nm (Desfougeres, Saint-Jalmes, Salonen, Vié, Beaufils, Pezennec, et al., 2011). BHA, TBHQ, and TBQ were purchased from Aladdin Chemical Reagent (Shanghai, China), and their stock solutions were prepared at a concentration of 2.0×10–3 mol·l–1 with anhydrous ethanol. All of the other materials and reagents were of analytical grade, and ultrapure water was used throughout the experiment. All stock solutions were prepared weekly and stored in the dark at 4 °C. 2.2. Fluorescence measurements Fluorescence measurements were performed using a Cary Eclipse fluorescence spectrophotometer (Varian, America) equipped with 1.0 cm quartz cells according to our previous report (Wu, Yan, Wang, Wang, & Li, 2015). Prior to fluorescence 4

measurements, the solutions were vortex-mixed and maintained for 1 h in a thermostat water bath at 25 °C, 31 °C and 37 °C. An excitation wavelength of 295 nm was employed, and emission spectra were recorded from 300 nm to 500 nm, with widths of excitation and emission slits set at 5 and 10 nm, respectively. All fluorescence intensities used were corrected to decrease the inner filter effect via the previously reported method (Cahyana & Gordon, 2013). The three-dimensional (3D) fluorescence spectra of lysozyme (2.0×10–6 mol·l–1) and of the lysozyme–ligand complexes (molar ratio, 1:10) were obtained at an excitation wavelength range of 200 nm to 400 nm at 5 nm increments (Jash & Kumar, 2014). The emission spectra were also monitored between 200 and 500 nm. 2.3. CD spectroscopy Circular dichroism (CD) spectra were collected using a CD spectrometer (Model 400, AVIV, USA) at 25 °C with a 2 mm path length cell in nitrogen atmosphere. The spectra of lysozyme and its ligand complex were obtained from 260 nm to 190 nm wavelengths with a 1 nm step size, 1 nm band width, and 0.5 s response time (Liang, Liu, Qi, Su, Yu, Wang, et al., 2013; Wu, Yan, Wang, Wang, & Li, 2015). Each CD spectrum was the average of three scans. 2.4. Molecular docking All docking simulations were performed using AutoDock Version 4.2.5.1 program package and AutoDock Tools (ADT) Version 1.5.6 to identify the potential ligand binding sites. Lamarckian genetic algorithm (LGA) (Morris, Goodsell, 5

Halliday, Huey, Hart, Belew, et al., 1998) implemented in AutoDock was applied to estimate the possible conformations of the ligand–protein complex. The available lysozyme crystal structure used in the docking studies was obtained from the RCSB Protein Data Bank (PDB ID: 2LYZ). For the preparation of the receptor (lysozyme), water molecules were removed, polar hydrogen atoms were added, and Kollman united atom partial charges were assigned to lysozyme by using ADT. The 3D structures of the ligands (BHA, TBHQ, and TBQ) were generated with the use of ChemBioOffice Version 11.0 and optimized using the Austin model-1 (AM1) method (Rocha, Freire, Simas, & Stewart, 2006). Docking was conducted by setting the grid box size at 78 Å × 66 Å × 78 Å along the x, y, and z axes, thereby covering the whole lysozyme with a grid spacing of 0.492 Å. AutoGrid was then run to generate the grid map of the different ligand and receptor atoms (Balachandran, Kumar, Arun, Duraipandiyan, Sundaram, Vijayakumar, et al., 2014). After the grid map was generated, ligand flexible docking simulations were performed with 200 runs and 2.5×106 energy evaluations. A total of 27000 generations and a genetic algorithm population of 150 individuals were used to locate the optimum binding site. In addition, cluster analysis was performed on the docked results (Wu, Zhai, Yan, Xu, Wang, Li, et al., 2015). The result with the lowest docking energy analysis in cluster rank 1 was used for further analysis. For visualization of the docked conformations, Discovery Studio 3.1 (State Key Laboratory of Biotherapy, Sichuan University, China) software package was used. 2.5. Statistical analysis 6

In binding studies (fluorescence measurements), all assays were conducted in triplicate. The maximum experimental error in the measurements was 5% and the mean values, standard deviations, and statistical differences were estimated using analysis of variance. Data processing and analyses were performed using the OriginPro software (OriginLab Corporation, Northampton, MA). 3. Results and discussion 3.1. Fluorescence quenching analysis For macromolecules, fluorescence measurements can provide information on the binding properties of small molecule substances to protein (Kandagal, Seetharamappa, Ashoka, Shaikh, & Manjunatha, 2006). Lysozyme is a multi-tryptophan protein that contains six tryptophan (Trp) residues. When lysozyme interacts with other compounds, its intrinsic fluorescence, which is mainly caused by Trp 62, Trp 63, and Trp 108, often changes with ligand concentration (Ibrahim, Matsuzaki, & Aoki, 2001). Such a response would lead to a decrease in intensity and often occurs in conformational transitions, association, substrate binding, or denaturation, which is also called quenching (Lakowicz & Masters, 2008). Fluorescence quenching may either be dynamic, which is caused by diffusion, or static, which is caused by ground-state complex formation. The quenching mechanisms can be distinguished by the differences in temperature-dependent behaviour and described by the well-known Stern–Volmer equation (Valeur & Berberan-Santos, 2012):

F0 / F = 1 + K SV [ Q]

(2)

where F0 and F are the steady-state of the fluorescence intensities in the absence and 7

presence of a quencher, and [Q] is the quencher concentration. Ksv, which is determined by the linear regression of the plot of F0/F against [Q], is the Stern–Volmer quenching constant. The fluorescence spectral changes of lysozyme with different amounts of BHA (A), TBHQ (B), and TBQ (C) following an excitation at 295 nm, and their Stern–Volmer curves at 25 °C are illustrated in Fig 1. When the lysozyme was titrated with different amounts of BHA, TBHQ, and TBQ, an evident shrink of the fluorescence signal and a slight red shift at the maximum lysozyme wavelength were observed. Quenching was strong in the case of BHA and TBQ, whereas with the addition of TBHQ, the extent of quenching lessened. The tertiary structure of lysozyme, mainly the microenvironment of Trp residues, may have been changed by the binding behaviour of the three compounds. To confirm the quenching mechanism, quantitative analysis was carried out, and the results show good linearity of the F0/F plots for the lysozyme with various concentrations of ligands at different temperatures. The

obtained

Ksv

values

were

(1.215±0.0004)×104

(25 °C,

R2=0.9918),

(1.193±0.0012)×104 (31 °C, R2=0.9799), and (1.155±0.006)×104 L·mol–1 (37 °C, R2=0.9930)

for

BHA;

(1.989±0.007)×104 (25 °C,

R2=0.9870),

(1.921±0.0005)×104 (31 °C, R2=0.9903), and (1.892±0.0009)×10 4 L·mol–1 (37 °C, R2=0.9820)

for

TBHQ;

(9.459±0.007)×104

(25 °C,

R2=0.9855),

(9.211±0.0008)×104 (31 °C, R2=0.9811), and (8.845±0.0003)×104 L·mol–1 (37 °C, R2 =0.9900) for TBQ. The decrease in Ksv with increasing temperature indicated that the quenching mechanism of the interaction between BHA/TBHQ/TBQ and lysozyme 8

was initiated by complex formation rather than by dynamic collision (Lakowicz & Masters, 2008). 3.2. Binding property The binding constant (Ka) and number (n) of bound ligands to lysozyme were determined by plotting the double-logarithm regression curve of the fluorescence data by using the modified Stern–Volmer equation (Valeur & Berberan-Santos, 2012; Wu, Yan, Wang, Wang, & Li, 2015):

log( F0 − F ) / F = log K a + n log [ Q]

(3)

A regression curve could be derived using Eq. (3). The Ka (intercept) and n (slope) values are shown in Table 1. The magnitude of the values of Ka indicates that moderate interaction of BHA or its metabolites with lysozyme occurs. A larger Ka implies that the lysozyme–ligand complex is more stable. The order of binding affinity of lysozyme for all test compounds is: BHA > TBQ > TBHQ. With increasing temperature, the binding abilities of these three systems decreased. 3.3. Thermodynamic parameters and type of binding force In general, the essence of most protein–ligand recognition is non-covalent interactions, such as hydrophobic forces, van der Waals interactions, electrostatic interactions, or hydrogen bonds. These bonds are immediately mutable and are thereby amenable to standard equilibrium thermodynamic analysis (Peng, Ding, Peng, Jiang, & Zhang, 2013). Intermolecular forces can be explained by calculating the thermodynamic parameters (free energy change (∆G), enthalpy change (∆H), and entropy change (∆S)) of the binding reaction, which can be obtained based on the 9

binding constants of different temperatures using the van’t Hoff equation (Eq. 4) and Gibbs–Helmhotz equation (Eq. 5) (Haynie, 2001): ln K a = −

∆H ∆S + RT R

(4) (5)

∆G = ∆H − T ∆S

where Ka is the associative binding constant at the corresponding temperature, and R is the gas constant. As seen in Table 1, the negative values for ∆G indicate that the binding of these three compounds with lysozyme occurred spontaneously. Based on the theory of Ross and Olsson (Ross & Subramanian, 1981; Olsson, Williams, Pitt, & Ladbury, 2008), the negative values of ∆H and ∆S confirm that both hydrogen bonds and van der Waals forces have a predominant function in the interaction between BHA/TBHQ/TBQ and lysozyme.

3.4. Conformation investigation During the binding process, the intermolecular forces that maintain the secondary and tertiary structures of lysozyme may be altered. To further determine the BHA, TBHQ, and TBQ-lysozyme binding mechanism regarding the conformational changes of the protein, CD measurements and 3D fluorescence spectra were obtained. 3D fluorescence spectroscopy is a common and useful technology that provides details on the structural changes in proteins (Zhang, Xu, Zhou, & Wang, 2011). As shown in Fig. 2 (A to D), the 3D fluorescence spectra of lysozyme (A), BHA–lysozyme (B), TBHQ–lysozyme (C), and TBQ–lysozyme (D) were investigated, and the related characteristic parameters are listed in Table 2. Peaks a and b represent the first-order Rayleigh scattering peak (λex=λem) and the second-order Rayleigh 10

scattering peak (λex=2λem), respectively. Peak I exhibits the spectral characteristic of Trp and Tyr residues, and peak II refers to the fluorescence characteristics of the polypeptide backbone structures (Li, Zhang, Xu, & Ji, 2011). The addition of BHA/TBHQ/TBQ to the lysozyme solution produced a decrease, to differing degrees, in intensity along with a slight red shift of the maximum emission wavelength in peaks I and II. As seen in Table 2, BHA, TBHQ and TBQ decreased the fluorescence intensity of peak I to 69.1, 89.3, and 81.8%, whereas they decreased that of peak II to 82.6, 90.0, and 75.1%, respectively. Analysis of the intensity changes and peak shifts indicate that the binding of BHA or its metabolites to lysozyme induced conformational and microenvironmental changes of the protein. (Ghosh, Pandey, Sen, Tripathy, & Dasgupta, 2013; Lakowicz & Masters, 2008). Further evidence of the conformational changes of lysozyme upon the addition of BHA/TBHQ/TBQ was provided by CD spectra. CD is a universally acknowledged technique used for the structural characterization of proteins (Sreerama, Venyaminov, & Woody, 1999). CD spectroscopy provides rapid determination of protein secondary structure and fast assessment of the conformational changes caused by ligand addition (Kelly, Jess, & Price, 2005; Siligardi, Hussain, Patching, & Phillips-Jones, 2014). Figure 3 shows that the CD spectra of lysozyme exhibited a remarkably negative band in the far-UV region at 208 nm and a slightly negative band at 222 nm, which were mainly attributed to n→π* transfers of the peptide bond and characteristic features of the α-helix structure of lysozyme (Shanmugaraj, Anandakumar, & Ilanchelian, 2015). These ellipticities for α-helix content of lysozyme increased with the addition of 11

different ligands, and the changes induced by the compounds follow the order: BHA > TBQ > TBHQ. The different effects of these compounds on the α-helix content of lysozyme may be associated with binding affinity of the compounds toward lysozyme. The secondary structure is closely related to the biological activity of proteins, and the results indicated that BHA or its metabolites potentially adopt a more compact conformation in lysozyme.

3.5. Molecular docking To further substantiate the interaction between lysozyme and the model compounds, the complementary application of the molecular model was applied using the AutoDock program package. The AutoDock strategy was used to search the entire surface of the protein for binding sites that simultaneously optimize the conformations of the peptides (Morris, Huey, Lindstrom, Sanner, Belew, Goodsell, et al., 2009). According to X-ray diffraction measurement (Blake, Koenig, Mair, North, Phillips, & Sarma, 1965), lysozyme is a single, nonglycosylated polypeptide chain of 129 amino acid residues folded into two domains that are functional for the active site cleft. To promote visual understanding of the positions, a total of 11, 16, and 13 multimember conformational clusters were gathered from 200 docking runs for BHA, TBHQ, and TBQ, respectively. Optimally, the highest populated cluster contained over half of the conformations obtained during the docking procedure with the lowest binding energy as shown in Fig. 4 (A to C). Data derived from the docking of BHA (A), TBHQ (B) and TBQ (C) molecules to lysozyme showed different modes of interactions. Figure 4A shows that the BHA 12

molecule was mainly surrounded by the residues Gln57, Trp62, Trp63, Ile58, Ala107, Asp52, Trp108, Ile98, Leu56, and Asn59 with a binding energy of –4.26 kcal·mol–1, which are active amino acids for the lysozyme–BHA interaction. The locations of TBHQ and TBQ were characterized by favourable binding energies of −3.65 and −4.07 kcal·mol–1. In all three systems, hydrogen bond networks contributed to 3D space position changes in ligands and adapted to the shape of the pocket of lysozyme and stabilized the docking conformation. A hydrogen bond was found 1.66 Å between TBQ oxygen and residue Trp63. Trp63 and Gln57 were in a suitable position to form two hydrogen bonds with BHA, with distances of 1.81 and 2.02 Å, respectively. The residues of lysozyme (Leu56, Ala107, and Trp108) in the binding pocket interact with TBHQ to also form three hydrogen bonds with distances of 2.07 Å (Leu 56), 1.88 Å (Ala 107) and 1.79 Å (Trp 108), respectively, whereas Asp52, Val109, and Ile98 favoured van der Waals forces. These forces stabilized the docking conformation and were consistent with the binding mode obtained from thermodynamic parameters. Furthermore, these results provided a good structural basis for explaining the efficient fluorescence quenching of lysozyme emission in the presence of BHA or its metabolites. 4. Conclusions The binding properties of BHA, TBHQ, and TBQ to lysozyme were characterized via multispectroscopic analyses and computer-aided molecular modeling studies. The results reveal that BHA and its metabolites can bind to lysozyme through static quenching and the binding affinity follows the order: BHA > 13

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423-429. Wu, D., Zhai, Y., Yan, J., Xu, K., Wang, Q., Li, Y., & Li, H. (2015). Binding mechanism of tauroursodeoxycholic acid to human serum albumin: insights from NMR relaxation and docking simulations. RSC Advances, 5(15), 11036-11042. Zhang, H. M., Xu, Y. Q., Zhou, Q. H., & Wang, Y. Q. (2011). Investigation of the interaction between chlorophenols and lysozyme in solution. Journal of

Photochemistry and Photobiology B: Biology, 104(3), 405-413.

Figure captions: Fig. 1. Effect of BHA (A), TBHQ (B), and TBQ (C) on the fluorescence spectra of lysozyme (pH 6.5, T ＝25 °C and λem ＝ 295 nm). The 2.0×10 –6 mol·l–1 lysozyme solution with 0.0×10 –6, 2.0×10–6, 4.0×10–6, 6.0×10–6, 8.0×10–6, 1.0×10 –5, and 1.2×10–5 mol·l–1 BHA (TBHQ or TBQ) from up and downward, respectively. The inset shows Stern–Volmer plots for lysozyme fluorescence quenching caused by BHA/TBHQ/TBQ at the above conditions. All data were corrected for quencher fluorescence, and each data point was the mean of three independent determinations ± SD ranging from 0.53 to 1.76%. Fig. 2. 3D fluorescence spectra of lysozyme (A),

BHA–lysozyme (B),

TBHQ–lysozyme (C), and TBQ–lysozyme (D). The concentration of lysozyme was 2.0×10–6 mol·l–1. The molar ratio of lysozyme to ligand was 1:1. Fig. 3. CD spectra of lysozyme in the absence (black) and presence of BHA (green), TBHQ (red), and TBQ (blue). The concentration of lysozyme is 1.0×10–6 mol·l–1, and 20

the concentrations of BHA, TBHQ, and TBQ were all 2.0×10–6 mol·l–1. Fig. 4. 3D view and schematic diagram generated using the 2D diagram feature of Accelrys Discovery Studio 3.1 show the binding modes between BHA (A), TBHQ (B), or TBQ (C) and lysozyme. The pink circles represent the residues participating in hydrogen bonds, charge, or polar interactions. The green circles are residues participating in van der Waals interactions. The light blue circle surrounding a given residue/atom denotes its solvent-accessible surface.

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Table 1. Modified Stern–Volmer binding constant (Ka), number of binding sites (n) and relative thermodynamic parameters for the association of lysozyme with BHA, TBHQ, and TBQ at different temperatures. n

Ra

(°C)

Ka (L∙mol-1)

ΔG (kJ∙mol-1)

ΔH (kJ∙mo-1)

ΔS (J∙mol-1∙K-1)

BHA

25 31 37

(9.488±0.013) ×105 (6.741±0.054) ×105 (4.860±0.106) ×105

1.0670±0.0901 1.2002±0.0709 1.1285±0.1317

0.9957 0.9926 0.9918

-34.10 -33.92 -33.75

-42.82

-29.28

TBHQ

25 31 37

(3.548±0.052) ×104 (2.225±0.098) ×104 (1.422±0.114) ×104

1.0520±0.1054 1.0151±0.0869 0.9984±0.1509

0.9935 0.9921 0.9909

-25.96 -25.30 -24.65

-58.51

-109.23

TBQ

25 31 37

(5.455±0.061) ×105 (4.660±0.046) ×105 (4.065±0.039) ×105

1.1615±0.1178 1.0088±0.1264 1.0342±0.0986

0.9917 0.9923 0.9939

-32.73 -33.00 -33.29

-18.82

46.67

Compounds

a

T

Correlation coefficient for the Ka values.

Table 2. 3D fluorescence spectral characteristic parameters of lysozyme, lysozyme–BHA, lysozyme–TBHQ, and lysozyme–TBQ. System

Peak No.

Peak position [Ex/Em (nm/nm)]

Intensity F (a.u.)

lysozyme

I II I II I II I

280/333 225/333 280/344 225/337 280/334 225/336 280/340

525.79 513.53 363.31 424.38 469.85 462.17 430.40

II

225/336

385.77

lysozyme–BHA lysozyme–TBHQ lysozyme–TBQ

Highlights: BHA and its metabolites interact with lysozyme at different extents. Binding is predominantly the result of hydrogen bond and van der Waals force. Structural changes of lysozyme have been demonstrated. Computational studies of the test compounds with lysozyme substantiate the experimental findings.

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