Research Article Received: 19 June 2014,
Revised: 24 July 2014,
Accepted: 18 September 2014,
Published online in Wiley Online Library: 12 February 2015
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2437
Characterization of the binding of 2-mercaptobenzimidazole to bovine serum albumin Yue Tenga*, Luyi Zoua, Ming Huanga and Wansong Zongb 2-Mercaptobenzimidazole (MBI) is widely utilized as a corrosion inhibitor, copper-plating brightener and rubber accelerator. The residue of MBI in the environment is potentially harmful to human health. In this article, the interaction of MBI with bovine serum albumin (BSA) was explored using spectroscopic and molecular docking methods under physiological conditions. The positively charged MBI can spontaneously bind with the negatively charged BSA through electrostatic forces with one binding site. The site marker competition experiments and the molecular docking study revealed that MBI bound into site II (subdomain IIIA) of BSA, which further led to some secondary structure and microenvironmental changes of BSA. This work provides useful information on understanding the toxicological actions of MBI at the molecular level. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: serum albumin; 2-mercaptobenzimidazole; multi-spectroscopic techniques; displacement experiments; molecular docking
INTRODUCTION
232
Serum albumins are the most abundant extracellular proteins in the mammalian circulatory system and account for about 50–60% of the total plasma proteins (Carter and Ho, 1994). Serum albumins perform many critical physiological functions. For instance, they are responsible for the maintenance of osmotic blood pressure and contribute to keeping blood pH (Basu and Kumar, 2014). However, the most important physiological role of albumins involves serving as transport vehicles for a variety of endogenous and exogenous compounds (Neelam et al., 2010). Bovine serum albumin (BSA) has become one of the most extensively studied proteins because of its low cost, availability and structural similarity with human serum albumin (HSA) (MacManus-Spencer et al., 2010; Lin et al., 2013). BSA is a 582-residue monomer and contains three homologous domains (namely, I, II, III), each of which is composed of two subdomains (denoted by A and B) (Jana et al., 2012). The binding of toxins with BSA has toxicological importance, because the degree and time of action in the body affect the duration and intensity of their effects (Guo et al., 2014; Wang et al., 2014). 2-Mercaptobenzimidazole (MBI) is an important member of the thioureylene compound family that is administered as a corrosion inhibitor (Finsgar, 2013a, 2013b), copper-plating brightener (Cheng et al., 1999), rubber accelerator and/or antioxidant (Sakemi et al., 2002). Although the usability of MBI is indisputable, it is known as a toxic and poorly biodegradable pollutant (Rastegarzadeh and Rezaei, 2013). Thus, wide use of MBI leads to an increase in the possibility of its exposure to organisms. Previous studies reported that MBI could be found as a contamination source in rubber plant waste water (Khramov et al., 2005), rivers (Jungclaus et al., 1978), street runoff (Spies et al., 1987) and some drugs contaminated with MBI from the rubber plunger seals of syringes and/or drug packing containers (Airaudo et al., 1990).
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The toxic effects of MBI on experimental animals have been reported. MBI had potent antithyroid toxicity in rats on a 28-day repeated oral dosing (Kawasaki et al., 1998). An inhalation toxicity of MBI on rats revealed that exposure to MBI resulted in increased thyroid weight, thyroid follicular cell hyperplasia, reduced triiodothyronine and thyroxine levels (Gaworski et al., 1991). Doerge reported that thioureylene antithyroid compounds blocked the biosynthesis of thyroxine (T4) by inhibiting thyroid peroxidase (TPX) (Doerge, 1986). Yamano et al. (1995) explored the adverse effects of MBI on pregnant rats and their fetuses and observed major fetal malformations. They concluded that maternal toxicity preceded fetal toxicity. However, little information is available on the molecular interactions of the effect of MBI on the carrier protein BSA. In this work, we aimed to explore the interaction mechanism of MBI with BSA by integrating the application of spectroscopic and molecular docking methods. We studied the binding parameters of the interaction. The precise binding site of MBI on BSA was investigated in detail. The effect of MBI on the conformation of BSA was also explored. This study will be helpful to evaluate the toxicology of MBI and understand its effects on the function of protein during the blood transportation process.
* Correspondence to: Y. Teng, School of Environmental and Civil Engineering, Jiangnan University, Wuxi 214122, China. E-mail: [email protected] a Y. Teng, L. Zou, M. Huang School of Environmental and Civil Engineering, Jiangnan University, 1800# Lihu Avenue, Wuxi 214122, China b W. Zong College of Population, Resources and Environment, Shandong Normal University, 88# Wenhua Road, Jinan 250014, China
Copyright © 2015 John Wiley & Sons, Ltd.
BINDING OF 2-MERCAPTOBENZIMIDAZOLE TO BOVINE SERUM ALBUMIN 1.0-cm quartz cells. Slit width was set at 2.0 nm. The wavelength range was 200–280 nm.
EXPERIMENTAL METHODS Reagents Bovine serum albumin (BSA) and 2-mercaptobenzimidazole (MBI) were obtained from Sinopharm Chemical Reagent Co., Ltd. MBI was dissolved with ultrapure water as a stock solution, 1.0 × 10-3 mol l-1. Phenylbutazone (PB) and flufenamic acid (FA) from Tokyo Chemical Industry Co. Ltd. and digitoxin (Dig) form Dr. Ehrenstorfer GmbH were dissolved in ethanol to form a 1.0 × 10-3-mol l-1 solution, which was used to determine the binding sites of MBI on BSA. A 0.2-mol l-1 mixture of phosphate buffer (mixture of NaH2PO4 · 2H2O and Na2HPO4 · 12H2O, pH = 7.4) was used to control the pH. All other reagents were of analytical grade and purchased from standard reagent suppliers. Ultrapure water (18.25 MΩ) was used throughout the experiments. Apparatus and measurements
Synchronous fluorescence spectra of BSA in the absence and presence of MBI were measured (Δλ = 15 nm, λem = 280–335 nm and Δλ = 60 nm, λem = 310–370 nm, respectively) by an RF-5301PC fluorescence spectrophotometer (Shimadzu Japan). The excitation and emission slit widths were set at 5 nm. Circular dichroism (CD) measurements CD spectra were made on a MOS-450/AF-CD Spectropolarimeter (Bio-Logic, France) in a 1.0-cm cell at room temperature. Bandwidth was 4 nm, and scanning speed was 1 nm/2 sec.
RESULTS AND DISCUSSION
Fluorescence measurements All fluorescence spectra were recorded on an RF-5301PC fluorescence spectrophotometer (Shimadzu Japan) with a 1-cm cell. The excitation wavelength was 280 nm. The excitation and emission slit widths were set at 5 nm. Displacement experiments The displacement experiments were performed using different site markers, namely, PB, FA and Dig for sites I, II and III, respectively, by keeping the concentrations of BSA and the markers constant at 3.0 × 10-7 mol l-1, and then gradually adding MBI (to give a final concentration of 5.0 × 10-5 mol l-1). Fluorescence quenching spectra were measured at 285 K over a range of 290–500 nm. The binding constants of the MBI–BSA system in the presence of above site markers were calculated by the fluorescence data. Molecule docking investigation Docking calculations were carried out using AutoDock 4.2 (developed by The Scripps Research Institute, USA) (Morris et al., 2009). Molecular Operating Environment (MOE) version 2007.0902 (developed by Chemical Computing Group Inc, Canada) was used to prepare the structure of MBI and obtain the energy-minimized conformation of MBI (Vilar et al., 2008). Docking calculations were carried out on a BSA protein model (PDB code 4F5S) (Bujacz, 2012; Kumari et al., 2014). With the aid of AutoDock, the ligand root of MBI was detected, and rotatable bonds were defined. All hydrogen atoms and compute gasteiger charges were added into the BSA protein model. To recognize the binding sites in BSA, blind docking was carried out, and grid maps of 126 × 126 × 126 Å grid points and 0.375 Å spacing were generated. Docking simulations were performed using the Lamarckian genetic algorithm search method. Each run of the docking experiment was set to terminate after a maximum of 250 000 energy evaluations, and the population size was set to 150. The conformation with the lowest binding free energy was used for further analysis. Ultraviolet-visible (UV-vis) absorption measurements
Characterization of the binding interaction of MBI with BSA by fluorescence measurements Fluorescence quenching Fluorescence methods have been widely used to study the interaction between ligands and proteins and can provide significant parameters such as the quenching mechanism, binding constants and binding sites. We utilized the technique to investigate the interaction between MBI and BSA. Fluorescence of BSA is mostly attributed by tryptophan (Try) residues. BSA has two Try residues: Try-134 residue is located on the surface of subdomain IB and Trp-212 residue within the hydrophobic binding pocket of subdomain IIA (Yang et al., 2014). In addition, the fluorescence of Try residue is very sensitive to the local environment, making it a good method for the detecting of protein conformational changes (Lynch and Dawson, 2008). The fluorescence spectra of BSA at various concentrations of MBI are shown in Figure 1. BSA exhibited a strong fluorescence emission peak at 342 nm. The fluorescence intensity of BSA decreased regularly with increasing amounts of MBI to BSA solution, which indicated that MBI can bind to BSA and alter the structure of BSA.
-
Figure 1. Effect of MBI on BSA fluorescence. Conditions: BSA: 3.0 × 10 7 -1 -5 -1 mol l ; MBI/(×10 mol l ): (a) 0, (b) 1, (c) 2, (d) 3, (e) 4 and (f) 5; pH 7.4; T = 285 K.
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The absorption spectra were recorded on a double-beam UV-6100 spectrophotometer (Mapada, China) equipped with
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Synchronous fluorescence measurements
Y. TENG ET AL. Because MBI can quench the intrinsic fluorescence of BSA, we further analyzed the quenching mechanism. Quenching mechanisms include static and dynamic quenching. For dynamic quenching, higher temperatures result in faster diffusion and larger amounts of collision quenching so that the quenching constant increases with increasing temperature. In contrast, increased temperature is likely to result in decreased stability of complexes and thus lower the value of the static quenching constants (Liu et al., 2014). To confirm the mechanism, the fluorescence quenching data were analyzed according to the Stern–Volmer equation (Lakowicz and Weber, 1973): F0 ¼ 1 þ K sv ½Q ¼ 1 þ k q τ 0 ½Q F
τ 0 is the fluorescence lifetime in the absence of quencher (the value of τ 0 is 10-8 sec) (Lakowicz and Weber, 1973). The Stern–Volmer curves of F0/F against [Q] at 285 and 310 K are shown in Figure 2. The KSV values for the MBI–BSA system were calculated from the slope of these plots and are listed in Table 1. The KSV values decreased with higher temperature. In addition, the maximum dynamic quenching constant kq of various quenchers is 2.0 × 1010 l mol-1 sec-1 (Ware, 1962). However, the values of kq at 285 and 310 K are far greater than 2.0 × 1010 l mol-1 sec-1. Therefore, the results indicated that the overall quenching was dominated by a static quenching mechanism forming a MBI–BSA complex.
(1)
where F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively. KSV is the Stern–Volmer quenching constant, [Q] is the concentration of the quencher, kq is the quenching rate constant of the biological macromolecule and
Binding parameters For the static quenching process, the number of binding sites (n) and the binding constant (Ka) can be obtained from the following formula (Ojha and Das, 2010):
lg
ðF 0 F Þ ¼ lgK a þ n lg½Q F
(2)
where F0, F and [Q] are the same as in Equation (1), Ka is the binding constant and n is the number of binding sites. The Ka and n values for the interaction of MBI with BSA could be calculated by the slopes of the static quenching equation log[(F0 - F)/F] versus log[MBI] as shown in Supplementary information Figure S1, and the calculated results are summarized in Table 2. The values of n are approximately equal to 1, indicating that there was a single binding site in BSA for MBI. For each temperature, the binding constant Ka is of the order of 104, indicating that a strong interaction existed between MBI and BSA. Even if a low concentration of MBI is present in the blood, MBI can easily interact with BSA. Determination of the binding forces Figure 2. Stern–Volmer plots for the quenching of BSA by MBI at 285 and 310 K.
Table 1. Stern-Volmer quenching constants for the interaction of MBI with BSA at 285 K and 310 K T (K) 285 310
KSV (×104 l mol-1)
kq (×10 l mol-1 sec-1)
Ra
S.D.b
2.23 2.07
2.23 2.07
0.9961 0.9984
0.0411 0.0244
12
a
R is the correlation coefficient. S.D. is the standard deviation for the KSV values.
b
In the binding process between small molecular ligands and proteins, there are four types of non-covalent forces, including hydrophobic forces, hydrogen bonds, van der Waals interactions and electrostatic forces. Ross and Subramanian (1981) summed up the thermodynamic laws to determine the type of binding force; that is, if ΔH○ > 0 and ΔS○ > 0, the main forces are hydrophobic interactions; if ΔH○ < 0 and ΔS○ < 0, van der Waals and hydrogen bond interactions play the main roles in the binding process; if ΔH○ < 0 and ΔS○ > 0, electrostatic effects are dominant. When the temperature range is not too wide, the enthalpy change (ΔH○) can be regarded as a constant and can be approximated from Equation (3). The free-energy change (ΔG○) and the entropy change (ΔS○) for the interaction were calculated based on thermodynamic Equations (4) and (5):
Table 2. Binding constants and relative thermodynamic parameters of the MBI–BSA system T (K)
Ka (×104 l mol-1)
n
Ra
ΔH○ (kJ mol-1)
ΔS○ (J mol-1 K-1)
ΔG○ (kJ mol-1)
285 310
9.72 5.94
1.15 1.11
0.9955 0.9995
-14.5
44.5 44.5
-27.2 -28.3
234
a
R is the correlation coefficient for the Ka values.
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BINDING OF 2-MERCAPTOBENZIMIDAZOLE TO BOVINE SERUM ALBUMIN ln
K2 K1
¼
1 1 T1 T2
○ ΔH R
(3)
○
ΔG ¼ RT lnK ○
○
ΔG ¼ ΔH TΔS
(4) ○
(5)
where K1 and K2 are the binding constants (analogous to Ka in Equation (2)) at T1 and T2, and R is the universal gas constant. The thermodynamic parameters of the MBI–BSA system are presented in Table 2. The negative ΔG○ at 285 and 310 K indicated that the binding process was spontaneous. Based on the above summary of Ross and Subramanian, the negative ΔH○ and positive ΔS○ demonstrated that electrostatic interactions were the predominant driving forces in the formation of the MBI–BSA complex. MBI is easily positively charged (Trachli et al., 2002; Finsgar, 2013a). Because the pH (7.4) we used in this experimental conditions is much greater than the isoelectric point of BSA (pH 4.7), BSA is negatively charged. Positively charged MBI can spontaneously interact with the negatively charged BSA through electrostatic forces, which is in accordance with the calculated results of thermodynamic parameters. Identification of the specific binding sites on BSA Displacement experiments BSA is a globular protein composed of three homologous domains (I, II, III). Each domain contains two subdomains (A and B). It has been proved that BSA has two principal binding sites, site I and site II, which are hydrophobic cavities located in subdomains IIA and IIIA (Chi and Liu, 2011). In order to locate the binding site of MBI in protein, displacement experiments were performed using different site competitors, phenylbutazone (PB), flufenamic acid (FA) and digitoxin (Dig) for sites I, II and III, respectively. The emission spectra of ternary mixture of BSA, site marker and MBI were recorded. The corresponding binding constant values were evaluated and were shown in Table 3. MBI was not significantly displaced by PB or by Dig, whereas the binding constant values decreased remarkably in the presence of FA. Hence, we concluded that MBI bound to site II of BSA, which is located in the hydrophobic pocket of subdomain IIIA. Molecular docking analysis To further define the exact binding site of MBI on BSA, a molecular docking program was carried out. The best energy ranked result is shown in Figure 3. The docking result (Figure 3A) exhibited that MBI bound to BSA on subdomain IIIA, which was exactly consistent with the site-competitive displacement experiments. The detailed docking results are presented in Figure 3C. The amino acid residues in the vicinity of this binding site were Tyr 400, Asn 404, Lys 524, Gln 525 and Leu 528. The essential driving forces of MBI binding of this
Figure 3. Docking results of the MBI and BSA system. (A) Binding site of MBI to BSA. Subdomains of BSA are in different colors. (B) 2D structure of MBI with atom numbers. (C) Detailed illustration of the binding between MBI and BSA. Hydrogen bond is depicted as red dashed lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
site were electrostatic interactions, and this result was in accordance with that discussed above. As shown in Figure 3C, a hydrogen bond existed between the H atom at position 12 of MBI and the OH atom on Tyr 400. Van der Waal interactions and hydrophobic forces also existed, but electrostatic forces played a major role in the binding of MBI to BSA.
Table 3. Effects of site probe on the binding constant of MBI on BSA K (without the site probe) (104 l mol-1)
7.02
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5.13
7.27
Conformational changes of BSA induced by MBI UV-vis absorption spectroscopy UV-vis absorption spectroscopy can be applied to investigate protein structural changes and protein–ligand complex formation.
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9.72
K (with PB) K (with FA) K (with Dig) (104 l mol-1) (104 l mol-1) (104 l mol-1)
Y. TENG ET AL. We recorded the UV-vis absorption spectra of BSA with various amounts of MBI (Figure 4). BSA has a strong absorption peak at about 210 nm, which is associated with the framework conformation of BSA (Yang et al., 2009). With increasing amounts of MBI added to BSA, the intensity of the absorption peak of BSA decreased with a slight red shift (from 210 to 215 nm), indicating that the bind of MBI with BSA resulted in the changes in the BSA conformation (Hu et al., 2004). Synchronous fluorescence Synchronous fluorescence measurements can provide direct information about the change of the molecular microenvironment around the fluorophores such as tryptophan (Try) and tyrosine (Tyr). The shift in the position of the synchronous fluorescence peak is closely related to the changes of the polarity around the fluorophores. When the intervals (Δλ) are set at 15 or 60 nm, synchronous fluorescence can give characteristic information of Tyr or Try, respectively (Guo et al., 2008). With the increasing concentration of MBI, the Tyr peak (in Figure 5A) and the Try peak (in Figure 5B) had obvious blue shifts, revealing that the conformation of BSA was changed such that the polarities around the Tyr and Try residues decreased and the hydrophobicity increased. As shown in Figure 6, the slope was higher when Δλ was 15 nm, indicating that the microenvironment of Tyr was influenced more by MBI than that of Try. Based on the docking results, MBI could bind with Tyr 400 of BSA through a hydrogen bond, which is identical with the results of Figure 6. Circular dichroism In order to further confirm the possible secondary structure changes, CD spectra of BSA in the absence and presence of MBI were recorded and shown in Figure 7. The four secondary structure contents of α-helix, β-sheet, turns and unordered were analyzed by using the CDPro software package (Table 4) (Sreerama and Woody, 2004). BSA has the secondary structures of 43.6% α-helix, 17.2% β-sheet, 16.1% turns and 24.3% unordered. With the addition of MBI to BSA (1:10), the α-helix decreased by 7.3%, the β-sheet
Figure 5. Synchronous fluorescence spectra of BSA (A) Δλ = 15 nm and -7 -1 -5 -1 (B) Δλ = 60 nm. Conditions: BSA: 3.0 × 10 mol l ; MBI/(×10 mol l ): (a) 0, (b) 1, (c) 2, (d) 3, (e) 4 and (f) 5; pH 7.4; T = 285 K.
Figure 6. Quenching of BSA synchronous fluorescence by MBI. Condi-7 -1 tions: BSA: 3.0 × 10 mol l ; (○) Δλ = 15 nm and (■) Δλ = 60 nm.
236
Figure 4. UV-vis spectra of BSA in the presence of different concentrations of MBI (versus the same concentration of MBI solution). Conditions: -6 -1 -5 -1 BSA: 2.5 × 10 mol l ; MBI/(×10 mol l ): (a) 0, (b) 1 and (c) 2; pH 7.4; T = 285 K.
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increased by 0.3%, the turns increased by 4.7% and the unordered increased by 4.1%. The decrease of α-helix content may affect the physiological functions of BSA. These results indicated that the
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BINDING OF 2-MERCAPTOBENZIMIDAZOLE TO BOVINE SERUM ALBUMIN binding of MBI caused some secondary structure changes in BSA. On the basis of the above experimental results, the binding of MBI to BSA induced conformational changes in BSA, and MBI had an obvious denaturing effect on BSA.
CONCLUSIONS
-7
-1
Figure 7. CD spectra of MBI–BSA system. Conditions: BSA: 2.0 × 10 mol l ; pH 7.4; T = 285 K.
Table 4. Effects of MBI on the percentage of secondary structural elements in BSA at 285 K Molar ratio of BSA to MBI 1:0 1:10
In this work, the interaction of MBI with BSA was investigated employing multiple spectroscopic and molecular docking methods under simulated physiological conditions in vitro. On the basis of spectroscopic results, the positively charged MBI can spontaneously bind with the negatively charged BSA with one binding site mainly through electrostatic forces to form the MBI–BSA complex. The exact conformation of MBI in the binding site was explored using sitecompetitive replacement experiments and molecular docking simulation. MBI bound with BSA in site II (subdomain IIIA). MBI triggered changes in the secondary structure and microenvironment of BSA as analyzed by the UV-vis absorption, synchronous fluorescence and CD spectra. This work provides basic information for clarifying the binding mechanisms of MBI with BSA and is helpful for understanding its effect on protein function during the blood transportation process and its toxicity in vivo.
Acknowledgements
Secondary structural elements in BSA α-Helix 43.6% 36.3%
β-Sheet
Turns
Unordered
17.2% 17.5%
16.1% 20.8%
24.3% 28.4%
The work is supported by the National Nature Science Foundation of China (NSFC, 21307043 and 21207082), and the Independent Research Project of Jiangnan University (JUSRP1032) is also acknowledged.
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J. Mol. Recognit. 2015; 28: 232–238
Revised: 24 July 2014,
Accepted: 18 September 2014,
Published online in Wiley Online Library: 12 February 2015
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2437
Characterization of the binding of 2-mercaptobenzimidazole to bovine serum albumin Yue Tenga*, Luyi Zoua, Ming Huanga and Wansong Zongb 2-Mercaptobenzimidazole (MBI) is widely utilized as a corrosion inhibitor, copper-plating brightener and rubber accelerator. The residue of MBI in the environment is potentially harmful to human health. In this article, the interaction of MBI with bovine serum albumin (BSA) was explored using spectroscopic and molecular docking methods under physiological conditions. The positively charged MBI can spontaneously bind with the negatively charged BSA through electrostatic forces with one binding site. The site marker competition experiments and the molecular docking study revealed that MBI bound into site II (subdomain IIIA) of BSA, which further led to some secondary structure and microenvironmental changes of BSA. This work provides useful information on understanding the toxicological actions of MBI at the molecular level. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: serum albumin; 2-mercaptobenzimidazole; multi-spectroscopic techniques; displacement experiments; molecular docking
INTRODUCTION
232
Serum albumins are the most abundant extracellular proteins in the mammalian circulatory system and account for about 50–60% of the total plasma proteins (Carter and Ho, 1994). Serum albumins perform many critical physiological functions. For instance, they are responsible for the maintenance of osmotic blood pressure and contribute to keeping blood pH (Basu and Kumar, 2014). However, the most important physiological role of albumins involves serving as transport vehicles for a variety of endogenous and exogenous compounds (Neelam et al., 2010). Bovine serum albumin (BSA) has become one of the most extensively studied proteins because of its low cost, availability and structural similarity with human serum albumin (HSA) (MacManus-Spencer et al., 2010; Lin et al., 2013). BSA is a 582-residue monomer and contains three homologous domains (namely, I, II, III), each of which is composed of two subdomains (denoted by A and B) (Jana et al., 2012). The binding of toxins with BSA has toxicological importance, because the degree and time of action in the body affect the duration and intensity of their effects (Guo et al., 2014; Wang et al., 2014). 2-Mercaptobenzimidazole (MBI) is an important member of the thioureylene compound family that is administered as a corrosion inhibitor (Finsgar, 2013a, 2013b), copper-plating brightener (Cheng et al., 1999), rubber accelerator and/or antioxidant (Sakemi et al., 2002). Although the usability of MBI is indisputable, it is known as a toxic and poorly biodegradable pollutant (Rastegarzadeh and Rezaei, 2013). Thus, wide use of MBI leads to an increase in the possibility of its exposure to organisms. Previous studies reported that MBI could be found as a contamination source in rubber plant waste water (Khramov et al., 2005), rivers (Jungclaus et al., 1978), street runoff (Spies et al., 1987) and some drugs contaminated with MBI from the rubber plunger seals of syringes and/or drug packing containers (Airaudo et al., 1990).
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The toxic effects of MBI on experimental animals have been reported. MBI had potent antithyroid toxicity in rats on a 28-day repeated oral dosing (Kawasaki et al., 1998). An inhalation toxicity of MBI on rats revealed that exposure to MBI resulted in increased thyroid weight, thyroid follicular cell hyperplasia, reduced triiodothyronine and thyroxine levels (Gaworski et al., 1991). Doerge reported that thioureylene antithyroid compounds blocked the biosynthesis of thyroxine (T4) by inhibiting thyroid peroxidase (TPX) (Doerge, 1986). Yamano et al. (1995) explored the adverse effects of MBI on pregnant rats and their fetuses and observed major fetal malformations. They concluded that maternal toxicity preceded fetal toxicity. However, little information is available on the molecular interactions of the effect of MBI on the carrier protein BSA. In this work, we aimed to explore the interaction mechanism of MBI with BSA by integrating the application of spectroscopic and molecular docking methods. We studied the binding parameters of the interaction. The precise binding site of MBI on BSA was investigated in detail. The effect of MBI on the conformation of BSA was also explored. This study will be helpful to evaluate the toxicology of MBI and understand its effects on the function of protein during the blood transportation process.
* Correspondence to: Y. Teng, School of Environmental and Civil Engineering, Jiangnan University, Wuxi 214122, China. E-mail: [email protected] a Y. Teng, L. Zou, M. Huang School of Environmental and Civil Engineering, Jiangnan University, 1800# Lihu Avenue, Wuxi 214122, China b W. Zong College of Population, Resources and Environment, Shandong Normal University, 88# Wenhua Road, Jinan 250014, China
Copyright © 2015 John Wiley & Sons, Ltd.
BINDING OF 2-MERCAPTOBENZIMIDAZOLE TO BOVINE SERUM ALBUMIN 1.0-cm quartz cells. Slit width was set at 2.0 nm. The wavelength range was 200–280 nm.
EXPERIMENTAL METHODS Reagents Bovine serum albumin (BSA) and 2-mercaptobenzimidazole (MBI) were obtained from Sinopharm Chemical Reagent Co., Ltd. MBI was dissolved with ultrapure water as a stock solution, 1.0 × 10-3 mol l-1. Phenylbutazone (PB) and flufenamic acid (FA) from Tokyo Chemical Industry Co. Ltd. and digitoxin (Dig) form Dr. Ehrenstorfer GmbH were dissolved in ethanol to form a 1.0 × 10-3-mol l-1 solution, which was used to determine the binding sites of MBI on BSA. A 0.2-mol l-1 mixture of phosphate buffer (mixture of NaH2PO4 · 2H2O and Na2HPO4 · 12H2O, pH = 7.4) was used to control the pH. All other reagents were of analytical grade and purchased from standard reagent suppliers. Ultrapure water (18.25 MΩ) was used throughout the experiments. Apparatus and measurements
Synchronous fluorescence spectra of BSA in the absence and presence of MBI were measured (Δλ = 15 nm, λem = 280–335 nm and Δλ = 60 nm, λem = 310–370 nm, respectively) by an RF-5301PC fluorescence spectrophotometer (Shimadzu Japan). The excitation and emission slit widths were set at 5 nm. Circular dichroism (CD) measurements CD spectra were made on a MOS-450/AF-CD Spectropolarimeter (Bio-Logic, France) in a 1.0-cm cell at room temperature. Bandwidth was 4 nm, and scanning speed was 1 nm/2 sec.
RESULTS AND DISCUSSION
Fluorescence measurements All fluorescence spectra were recorded on an RF-5301PC fluorescence spectrophotometer (Shimadzu Japan) with a 1-cm cell. The excitation wavelength was 280 nm. The excitation and emission slit widths were set at 5 nm. Displacement experiments The displacement experiments were performed using different site markers, namely, PB, FA and Dig for sites I, II and III, respectively, by keeping the concentrations of BSA and the markers constant at 3.0 × 10-7 mol l-1, and then gradually adding MBI (to give a final concentration of 5.0 × 10-5 mol l-1). Fluorescence quenching spectra were measured at 285 K over a range of 290–500 nm. The binding constants of the MBI–BSA system in the presence of above site markers were calculated by the fluorescence data. Molecule docking investigation Docking calculations were carried out using AutoDock 4.2 (developed by The Scripps Research Institute, USA) (Morris et al., 2009). Molecular Operating Environment (MOE) version 2007.0902 (developed by Chemical Computing Group Inc, Canada) was used to prepare the structure of MBI and obtain the energy-minimized conformation of MBI (Vilar et al., 2008). Docking calculations were carried out on a BSA protein model (PDB code 4F5S) (Bujacz, 2012; Kumari et al., 2014). With the aid of AutoDock, the ligand root of MBI was detected, and rotatable bonds were defined. All hydrogen atoms and compute gasteiger charges were added into the BSA protein model. To recognize the binding sites in BSA, blind docking was carried out, and grid maps of 126 × 126 × 126 Å grid points and 0.375 Å spacing were generated. Docking simulations were performed using the Lamarckian genetic algorithm search method. Each run of the docking experiment was set to terminate after a maximum of 250 000 energy evaluations, and the population size was set to 150. The conformation with the lowest binding free energy was used for further analysis. Ultraviolet-visible (UV-vis) absorption measurements
Characterization of the binding interaction of MBI with BSA by fluorescence measurements Fluorescence quenching Fluorescence methods have been widely used to study the interaction between ligands and proteins and can provide significant parameters such as the quenching mechanism, binding constants and binding sites. We utilized the technique to investigate the interaction between MBI and BSA. Fluorescence of BSA is mostly attributed by tryptophan (Try) residues. BSA has two Try residues: Try-134 residue is located on the surface of subdomain IB and Trp-212 residue within the hydrophobic binding pocket of subdomain IIA (Yang et al., 2014). In addition, the fluorescence of Try residue is very sensitive to the local environment, making it a good method for the detecting of protein conformational changes (Lynch and Dawson, 2008). The fluorescence spectra of BSA at various concentrations of MBI are shown in Figure 1. BSA exhibited a strong fluorescence emission peak at 342 nm. The fluorescence intensity of BSA decreased regularly with increasing amounts of MBI to BSA solution, which indicated that MBI can bind to BSA and alter the structure of BSA.
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Figure 1. Effect of MBI on BSA fluorescence. Conditions: BSA: 3.0 × 10 7 -1 -5 -1 mol l ; MBI/(×10 mol l ): (a) 0, (b) 1, (c) 2, (d) 3, (e) 4 and (f) 5; pH 7.4; T = 285 K.
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The absorption spectra were recorded on a double-beam UV-6100 spectrophotometer (Mapada, China) equipped with
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Synchronous fluorescence measurements
Y. TENG ET AL. Because MBI can quench the intrinsic fluorescence of BSA, we further analyzed the quenching mechanism. Quenching mechanisms include static and dynamic quenching. For dynamic quenching, higher temperatures result in faster diffusion and larger amounts of collision quenching so that the quenching constant increases with increasing temperature. In contrast, increased temperature is likely to result in decreased stability of complexes and thus lower the value of the static quenching constants (Liu et al., 2014). To confirm the mechanism, the fluorescence quenching data were analyzed according to the Stern–Volmer equation (Lakowicz and Weber, 1973): F0 ¼ 1 þ K sv ½Q ¼ 1 þ k q τ 0 ½Q F
τ 0 is the fluorescence lifetime in the absence of quencher (the value of τ 0 is 10-8 sec) (Lakowicz and Weber, 1973). The Stern–Volmer curves of F0/F against [Q] at 285 and 310 K are shown in Figure 2. The KSV values for the MBI–BSA system were calculated from the slope of these plots and are listed in Table 1. The KSV values decreased with higher temperature. In addition, the maximum dynamic quenching constant kq of various quenchers is 2.0 × 1010 l mol-1 sec-1 (Ware, 1962). However, the values of kq at 285 and 310 K are far greater than 2.0 × 1010 l mol-1 sec-1. Therefore, the results indicated that the overall quenching was dominated by a static quenching mechanism forming a MBI–BSA complex.
(1)
where F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively. KSV is the Stern–Volmer quenching constant, [Q] is the concentration of the quencher, kq is the quenching rate constant of the biological macromolecule and
Binding parameters For the static quenching process, the number of binding sites (n) and the binding constant (Ka) can be obtained from the following formula (Ojha and Das, 2010):
lg
ðF 0 F Þ ¼ lgK a þ n lg½Q F
(2)
where F0, F and [Q] are the same as in Equation (1), Ka is the binding constant and n is the number of binding sites. The Ka and n values for the interaction of MBI with BSA could be calculated by the slopes of the static quenching equation log[(F0 - F)/F] versus log[MBI] as shown in Supplementary information Figure S1, and the calculated results are summarized in Table 2. The values of n are approximately equal to 1, indicating that there was a single binding site in BSA for MBI. For each temperature, the binding constant Ka is of the order of 104, indicating that a strong interaction existed between MBI and BSA. Even if a low concentration of MBI is present in the blood, MBI can easily interact with BSA. Determination of the binding forces Figure 2. Stern–Volmer plots for the quenching of BSA by MBI at 285 and 310 K.
Table 1. Stern-Volmer quenching constants for the interaction of MBI with BSA at 285 K and 310 K T (K) 285 310
KSV (×104 l mol-1)
kq (×10 l mol-1 sec-1)
Ra
S.D.b
2.23 2.07
2.23 2.07
0.9961 0.9984
0.0411 0.0244
12
a
R is the correlation coefficient. S.D. is the standard deviation for the KSV values.
b
In the binding process between small molecular ligands and proteins, there are four types of non-covalent forces, including hydrophobic forces, hydrogen bonds, van der Waals interactions and electrostatic forces. Ross and Subramanian (1981) summed up the thermodynamic laws to determine the type of binding force; that is, if ΔH○ > 0 and ΔS○ > 0, the main forces are hydrophobic interactions; if ΔH○ < 0 and ΔS○ < 0, van der Waals and hydrogen bond interactions play the main roles in the binding process; if ΔH○ < 0 and ΔS○ > 0, electrostatic effects are dominant. When the temperature range is not too wide, the enthalpy change (ΔH○) can be regarded as a constant and can be approximated from Equation (3). The free-energy change (ΔG○) and the entropy change (ΔS○) for the interaction were calculated based on thermodynamic Equations (4) and (5):
Table 2. Binding constants and relative thermodynamic parameters of the MBI–BSA system T (K)
Ka (×104 l mol-1)
n
Ra
ΔH○ (kJ mol-1)
ΔS○ (J mol-1 K-1)
ΔG○ (kJ mol-1)
285 310
9.72 5.94
1.15 1.11
0.9955 0.9995
-14.5
44.5 44.5
-27.2 -28.3
234
a
R is the correlation coefficient for the Ka values.
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BINDING OF 2-MERCAPTOBENZIMIDAZOLE TO BOVINE SERUM ALBUMIN ln
K2 K1
¼
1 1 T1 T2
○ ΔH R
(3)
○
ΔG ¼ RT lnK ○
○
ΔG ¼ ΔH TΔS
(4) ○
(5)
where K1 and K2 are the binding constants (analogous to Ka in Equation (2)) at T1 and T2, and R is the universal gas constant. The thermodynamic parameters of the MBI–BSA system are presented in Table 2. The negative ΔG○ at 285 and 310 K indicated that the binding process was spontaneous. Based on the above summary of Ross and Subramanian, the negative ΔH○ and positive ΔS○ demonstrated that electrostatic interactions were the predominant driving forces in the formation of the MBI–BSA complex. MBI is easily positively charged (Trachli et al., 2002; Finsgar, 2013a). Because the pH (7.4) we used in this experimental conditions is much greater than the isoelectric point of BSA (pH 4.7), BSA is negatively charged. Positively charged MBI can spontaneously interact with the negatively charged BSA through electrostatic forces, which is in accordance with the calculated results of thermodynamic parameters. Identification of the specific binding sites on BSA Displacement experiments BSA is a globular protein composed of three homologous domains (I, II, III). Each domain contains two subdomains (A and B). It has been proved that BSA has two principal binding sites, site I and site II, which are hydrophobic cavities located in subdomains IIA and IIIA (Chi and Liu, 2011). In order to locate the binding site of MBI in protein, displacement experiments were performed using different site competitors, phenylbutazone (PB), flufenamic acid (FA) and digitoxin (Dig) for sites I, II and III, respectively. The emission spectra of ternary mixture of BSA, site marker and MBI were recorded. The corresponding binding constant values were evaluated and were shown in Table 3. MBI was not significantly displaced by PB or by Dig, whereas the binding constant values decreased remarkably in the presence of FA. Hence, we concluded that MBI bound to site II of BSA, which is located in the hydrophobic pocket of subdomain IIIA. Molecular docking analysis To further define the exact binding site of MBI on BSA, a molecular docking program was carried out. The best energy ranked result is shown in Figure 3. The docking result (Figure 3A) exhibited that MBI bound to BSA on subdomain IIIA, which was exactly consistent with the site-competitive displacement experiments. The detailed docking results are presented in Figure 3C. The amino acid residues in the vicinity of this binding site were Tyr 400, Asn 404, Lys 524, Gln 525 and Leu 528. The essential driving forces of MBI binding of this
Figure 3. Docking results of the MBI and BSA system. (A) Binding site of MBI to BSA. Subdomains of BSA are in different colors. (B) 2D structure of MBI with atom numbers. (C) Detailed illustration of the binding between MBI and BSA. Hydrogen bond is depicted as red dashed lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
site were electrostatic interactions, and this result was in accordance with that discussed above. As shown in Figure 3C, a hydrogen bond existed between the H atom at position 12 of MBI and the OH atom on Tyr 400. Van der Waal interactions and hydrophobic forces also existed, but electrostatic forces played a major role in the binding of MBI to BSA.
Table 3. Effects of site probe on the binding constant of MBI on BSA K (without the site probe) (104 l mol-1)
7.02
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5.13
7.27
Conformational changes of BSA induced by MBI UV-vis absorption spectroscopy UV-vis absorption spectroscopy can be applied to investigate protein structural changes and protein–ligand complex formation.
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9.72
K (with PB) K (with FA) K (with Dig) (104 l mol-1) (104 l mol-1) (104 l mol-1)
Y. TENG ET AL. We recorded the UV-vis absorption spectra of BSA with various amounts of MBI (Figure 4). BSA has a strong absorption peak at about 210 nm, which is associated with the framework conformation of BSA (Yang et al., 2009). With increasing amounts of MBI added to BSA, the intensity of the absorption peak of BSA decreased with a slight red shift (from 210 to 215 nm), indicating that the bind of MBI with BSA resulted in the changes in the BSA conformation (Hu et al., 2004). Synchronous fluorescence Synchronous fluorescence measurements can provide direct information about the change of the molecular microenvironment around the fluorophores such as tryptophan (Try) and tyrosine (Tyr). The shift in the position of the synchronous fluorescence peak is closely related to the changes of the polarity around the fluorophores. When the intervals (Δλ) are set at 15 or 60 nm, synchronous fluorescence can give characteristic information of Tyr or Try, respectively (Guo et al., 2008). With the increasing concentration of MBI, the Tyr peak (in Figure 5A) and the Try peak (in Figure 5B) had obvious blue shifts, revealing that the conformation of BSA was changed such that the polarities around the Tyr and Try residues decreased and the hydrophobicity increased. As shown in Figure 6, the slope was higher when Δλ was 15 nm, indicating that the microenvironment of Tyr was influenced more by MBI than that of Try. Based on the docking results, MBI could bind with Tyr 400 of BSA through a hydrogen bond, which is identical with the results of Figure 6. Circular dichroism In order to further confirm the possible secondary structure changes, CD spectra of BSA in the absence and presence of MBI were recorded and shown in Figure 7. The four secondary structure contents of α-helix, β-sheet, turns and unordered were analyzed by using the CDPro software package (Table 4) (Sreerama and Woody, 2004). BSA has the secondary structures of 43.6% α-helix, 17.2% β-sheet, 16.1% turns and 24.3% unordered. With the addition of MBI to BSA (1:10), the α-helix decreased by 7.3%, the β-sheet
Figure 5. Synchronous fluorescence spectra of BSA (A) Δλ = 15 nm and -7 -1 -5 -1 (B) Δλ = 60 nm. Conditions: BSA: 3.0 × 10 mol l ; MBI/(×10 mol l ): (a) 0, (b) 1, (c) 2, (d) 3, (e) 4 and (f) 5; pH 7.4; T = 285 K.
Figure 6. Quenching of BSA synchronous fluorescence by MBI. Condi-7 -1 tions: BSA: 3.0 × 10 mol l ; (○) Δλ = 15 nm and (■) Δλ = 60 nm.
236
Figure 4. UV-vis spectra of BSA in the presence of different concentrations of MBI (versus the same concentration of MBI solution). Conditions: -6 -1 -5 -1 BSA: 2.5 × 10 mol l ; MBI/(×10 mol l ): (a) 0, (b) 1 and (c) 2; pH 7.4; T = 285 K.
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increased by 0.3%, the turns increased by 4.7% and the unordered increased by 4.1%. The decrease of α-helix content may affect the physiological functions of BSA. These results indicated that the
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BINDING OF 2-MERCAPTOBENZIMIDAZOLE TO BOVINE SERUM ALBUMIN binding of MBI caused some secondary structure changes in BSA. On the basis of the above experimental results, the binding of MBI to BSA induced conformational changes in BSA, and MBI had an obvious denaturing effect on BSA.
CONCLUSIONS
-7
-1
Figure 7. CD spectra of MBI–BSA system. Conditions: BSA: 2.0 × 10 mol l ; pH 7.4; T = 285 K.
Table 4. Effects of MBI on the percentage of secondary structural elements in BSA at 285 K Molar ratio of BSA to MBI 1:0 1:10
In this work, the interaction of MBI with BSA was investigated employing multiple spectroscopic and molecular docking methods under simulated physiological conditions in vitro. On the basis of spectroscopic results, the positively charged MBI can spontaneously bind with the negatively charged BSA with one binding site mainly through electrostatic forces to form the MBI–BSA complex. The exact conformation of MBI in the binding site was explored using sitecompetitive replacement experiments and molecular docking simulation. MBI bound with BSA in site II (subdomain IIIA). MBI triggered changes in the secondary structure and microenvironment of BSA as analyzed by the UV-vis absorption, synchronous fluorescence and CD spectra. This work provides basic information for clarifying the binding mechanisms of MBI with BSA and is helpful for understanding its effect on protein function during the blood transportation process and its toxicity in vivo.
Acknowledgements
Secondary structural elements in BSA α-Helix 43.6% 36.3%
β-Sheet
Turns
Unordered
17.2% 17.5%
16.1% 20.8%
24.3% 28.4%
The work is supported by the National Nature Science Foundation of China (NSFC, 21307043 and 21207082), and the Independent Research Project of Jiangnan University (JUSRP1032) is also acknowledged.
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