Figures
Abstract
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. In the present work, the toxic interaction of MBI with the important antioxidant enzyme copper-zinc superoxide dismutase (Cu/ZnSOD) was investigated using spectroscopic and molecular docking methods. MBI can interact with Cu/ZnSOD to form an MBI-Cu/ZnSOD complex. The binding constant, number of binding sites and thermodynamic parameters were measured, which indicated that MBI could spontaneously bind with Cu/ZnSOD with one binding site through hydrogen bonds and van der Waals forces. MBI bound into the Cu/ZnSOD interface of two subdomains, which caused some microenvironmental and secondary structure changes of Cu/ZnSOD and further resulted in the inhibition of Cu/ZnSOD activity. This work provides direct evidence at a molecular level to show that exposure to MBI could induce changes in the structure and function of the enzyme Cu/ZnSOD. The estimated methods in this work may be applied to probe molecular interactions of biomacromolecules and other pollutants and drugs.
Citation: Teng Y, Zou L, Huang M, Chen Y (2014) Molecular Interaction Mechanism between 2-Mercaptobenzimidazole and Copper-Zinc Superoxide Dismutase. PLoS ONE 9(8): e106003. https://doi.org/10.1371/journal.pone.0106003
Editor: Freddie Salsbury Jr, Wake Forest University, United States of America
Received: June 1, 2014; Accepted: July 31, 2014; Published: August 26, 2014
Copyright: © 2014 Teng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.
Funding: The work is supported by National Nature Science Foundation of China (NSFC 21307043, Yue Teng received the funding. URL: http://www.nsfc.gov.cn/), Independent Research Project of Jiangnan University (JUSRP1032, Yue Teng received the funding. URL: http://www.sytu.edu.cn/) is also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Aerobic metabolism would generate large quantities of reactive oxygen species (ROS) including superoxide radicals (O2·–), hydrogen peroxide (H2O2) and hydroxyl radicals (OH·), which readily react with various cellular components and cause widespread damage [1]–[4]. ROS has been identified as an important factor in cancers [5], [6], diabetes [7], aging [8], inflammation [9], arteriosclerosis [10] and sickle cell disease [11]. As the first line of antioxidant systems, superoxide dismutases (SODs), including copper-zinc superoxide dismutase (Cu/ZnSOD), manganese superoxide dismutase (MnSOD) and extracellular superoxide dismutase (ECSOD), play an essential role in the detoxification of ROS [12]. They can catalyze the dismutation of two O2·– anions to H2O2 and molecular oxygen [13]. Among the three families of SODs, Cu/ZnSOD is most commonly used by eukaryotes. The cytosols of virtually all eukaryotic cells contain the enzyme Cu/ZnSOD, which exists as a dimer [14]. When residues of contaminants in the environment enter an organism, they may interact with Cu/ZnSOD and affect the catalytic activity of Cu/ZnSOD in its tissues.
2-Mercaptobenzimidazole (MBI) is an important member of the thioureylene compound family that is applied in various industrial processes such as corrosion inhibition [15], [16], copper-plating brightening [17], rubber acceleration and/or antioxidation [18]. Although the usability of MBI is indisputable, it is known as a toxic and poorly biodegradable pollutant [19]. Therefore, wide use of MBI results in an increase in the probability of its exposure to organisms. Previous studies reported that MBI could be found as a contamination source in rubber plant waste water [20], rivers [21], street runoff [22] and some drugs (the latter can become contaminated from the MBI in the rubber plunger-seals of syringes and/or drug packing containers) [23].
The toxic effects of MBI on experimental animals have been reported. MBI had potent antithyroid toxicity in rats during a 28-day repeated oral dosing [24]. An inhalation toxicity of MBI on rats showed exposure to MBI caused increased thyroid weight, thyroid follicular cell hyperplasia, reduced triiodothyronine and thyroxine levels [25]. It was reported that thioureylene antithyroid compounds blocked the biosynthesis of thyroxine (T4) by inhibiting thyroid peroxidase (TPX) [26]. Yamano et al. investigated the adverse effects of MBI on pregnant rats and their fetuses and observed major fetal malformations. They concluded that maternal toxicity preceded fetal toxicity [27]. However, little work has been conducted that focus on the molecular interactions governing the effect of MBI on antioxidant enzymes. Thus, the purpose of this study was to understand the interaction mechanism of MBI with Cu/ZnSOD by integrating the binding parameters (association and binding forces) of the interaction and the effect of MBI on the conformation of Cu/ZnSOD by using multiple spectroscopic techniques and molecular modeling. The effects of MBI on the activity of Cu/ZnSOD were also investigated. This work provides basic data for clarifying the binding mechanisms of MBI with the enzyme Cu/ZnSOD and is helpful for understanding human health risk of MBI in vivo.
Materials and Methods
Reagents
Cu/ZnSOD from porcine erythrocytes was purchased from Biodee Biotechnology Co., Ltd. 2-Mercaptobenzimidazole (MBI), nitroblue tetrazolium (NBT), methionine, riboflavin and EDTA were obtained from Sinopharm Chemical Reagent Co., Ltd. A 0.2 molL−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
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.
Synchronous fluorescence spectra of Cu/ZnSOD in the absence and presence of MBI were measured (Δλ = 15 nm, λem = 280–330 nm and Δλ = 60 nm, λem = 310–380 nm, respectively) by an RF-5301PC fluorescence spectrophotometer (Shimadzu Japan). The excitation and emission slit widths were set at 5 nm.
UV-visible absorption measurements.
The absorption spectra were recorded on a double beam UV-6100 spectrophotometer (Mapada, China) equipped with 1.0 cm quartz cells. Slit width was set at 2.0 nm. The wavelength range was 200–260 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 s.
Molecule docking investigation.
Docking calculations were carried out using AutoDock 4.2 (developed by The Scripps Research Institute, USA) [28]. 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 [29]. As the crystal structure of Cu/ZnSOD from porcine erythrocytes is unavailable in Protein Data Bank and Cu/ZnSOD from porcine erythrocytes has 90.26% homological identity with human Cu/ZnSOD, the crystal structure of human Cu/ZnSOD (1HL4.pdb) was downloaded. A homodimer of the crystal structure was used for docking calculations. With the aid of AutoDock, the ligand root of MBI was detected and rotatable bonds were defined. All hydrogen atoms were added into the Cu/ZnSOD protein model. To recognize the binding sites in Cu/ZnSOD, 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 (LGA) 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.
Cu/ZnSOD enzyme inhibition assay.
MBI was dissolved in ultrapure water to form a 1.0×10−5 molL−1 stock solution. Different volumes (0, 30, 100 and 300 µL) of MBI solutions were taken in vials and each was mixed with 30 µL of 1 µmolL−1 Cu/ZnSOD solution and made up to 1 mL using ultrapure water. Then, the enzymes were incubated in MBI for 60, 120 or 240 min at 4°C. The activity of Cu/ZnSOD was measured by monitoring its ability to inhibit the photoreduction of nitroblue tetrazolium (NBT) [30]. Each 3 mL reaction mixture contained 20 mmolL−1 phosphate buffer (pH 7.8), 10 µmolL−1 EDTA, 13 mmolL−1 methionine, 75 mmolL−1 NBT, 2 µmolL−1 riboflavin and 300 µL of the enzyme mixture with different MBI concentrations. The final concentrations of MBI in the 3 mL reaction mixtures were 0, 30, 100 and 300 nmolL−1. Recording the increase in absorbance at 560 nm followed the production of blue formazan at room temperature [31], [32]. One unit of Cu/ZnSOD represents the amount inhibiting the photoreduction of NBT by 50%.
Results and Discussion
Characterization of the binding interaction of MBI with Cu/ZnSOD by fluorescence measurements
Fluorescence quenching.
Because changes of emission spectra can provide information about the structure and dynamics of macromolecules, fluorescence has been widely used to investigate the interaction between proteins and ligands. We utilized fluorescence quenching to study the interaction between Cu/ZnSOD and MBI.
The fluorescence spectra of Cu/ZnSOD at various concentrations of MBI are shown in Figure 1. The fluorescence intensity of Cu/ZnSOD decreased regularly with increasing MBI concentration. The fluorescence of Cu/ZnSOD can be quenched by MBI. It’s reported that Cu/ZnSOD can emit intrinsic fluorescence mainly due to tryptophan and tyrosine residues, and fluorescence quenching was related to structural changes of proteins [30], [33]. So, the fluorescence quenching of Cu/ZnSOD by MBI indicated that MBI may bind to and alter the structure of Cu/ZnSOD.
Conditions: Cu/ZnSOD: 3.0×10−6 molL−1; MBI/(×10−5 molL−1): (a) 0, (b) 1, (c) 2, (d) 3, (e) 4, (f) 5; (g): MBI (1×10−5 molL−1) + buffer (0.02 molL−1); pH 7.4; T = 293 K.
Quenching mechanisms include static and dynamic quenching. Because higher temperature results in larger diffusion coefficients, the dynamic quenching constants will increase with increasing temperature. In contrast, increased temperature is likely to result in decreased stability of complexes, and thus the static quenching constants are expected to decrease with increasing temperature [34]. To confirm the mechanism, the fluorescence quenching data were analyzed according to the Stern-Volmer equation [35]:(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 τ0 is the fluorescence lifetime in the absence of quencher.
Fluorescence data were analyzed according to F0/F versus [Q] at 293 and 310 K (Figure 2). Eq. (1) was applied to determine KSV (Table 1) by a linear regression plot of F0/F against [Q]. The value of kq was also obtained (the fluorescence lifetime of the biopolymer (τ0) is 10−8 s [36]). It can be seen from Table 2 that the KSV values decreased with increasing temperature. Moreover, the maximum dynamic quenching constant kq of various quenchers is 2.0×1010 Lmol−1s−1 [37]. However, the values of kq at 293 and 310 K are greater than 2.0×1010 Lmol−1s−1. The results indicated that the overall quenching was dominated by a static quenching mechanism, and in the process a MBI-Cu/ZnSOD complex was formed.
Binding parameters.
For the static quenching interaction, when small ligands bind independently to a set of equivalent sites on a macromolecule, the number of binding sites (n) and the binding constant (Ka) can be obtained from the following formula [38]:(2)where F0, F and [Q] are the same as in Eq. (1), Ka is the binding constant and n is the number of binding sites. Ka and n can be calculated by plotting log[(F0–F)/F] versus log[MBI] as shown in Figure S1. The values of n and Ka were calculated and shown in Table 2. The number of binding sites n is approximately equal to 1, indicating that there was one binding site in Cu/ZnSOD for MBI during their interaction. Although the values of Ka decreased with an increase temperature, the value of Ka in 310 K (310 K is very close to body temperature) was of the order of 104, indicating that a strong interaction existed between MBI and Cu/ZnSOD. Even if a low concentration of MBI is present in organs, MBI can easily interact with Cu/ZnSOD.
Determination of the binding forces.
The acting forces between small molecular ligands and biomoleculers may involve hydrophobic forces, hydrogen bonds, van der Waals’ interactions and electrostatic forces. When the temperature range is small, the enthalpy change (ΔH°) can be considered as a constant and can be approximated from Eq. (3). The free-energy change (ΔG°) and the entropy change (ΔS°) for the interaction were calculated based on Eq. (4) and (5):(3)
(4)
(5)where K1 and K2 are the binding constants (analogous to Ka in Eq. (2)) at T1 and T2, and R is the universal gas constant. Ross and Subramanian [39] summed up the thermodynamic laws for estimating the type of binding force. That is, if ΔH°<0 and ΔS°<0, van der Waals and hydrogen bond interactions play the main roles in the binding reaction; if ΔH°>0 and ΔS°>0, hydrophobic interactions are dominant; if ΔH°<0 and ΔS°>0, the main forces are electrostatic effects. The thermodynamic parameters of the MBI-Cu/ZnSOD system were listed in Table 3. The ΔG° at 293 and 310 K are negative, indicating that the interaction process is spontaneous. Based on the above summary of Ross and Subramanian, the negative ΔH° and ΔS° mean that hydrogen bonds and van der Waals forces played major roles in the formation of the MBI-Cu/ZnSOD complex.
Identification of the specific binding sites on Cu/ZnSOD
We employed the molecular docking method to identify the binding site of MBI. The exact binding site of MBI on Cu/ZnSOD with the lowest binding free energy is shown in Figure 3A. MBI bound into the interface of the two subdomains of Cu/ZnSOD. The detailed docking results are presented in Figure 3C, and the distances are listed in Table 3. The amino acid residues in the vicinity of this binding site were Gly 51, Asp 52, Asn 53, Cys 146, Gly 147 and Val 148 of chain A and Val 5, Cys 6, Val 7, Gly 147, Val 148 and Gly 150 of chain B. The essential driving forces of the MBI binding of this site were hydrogen bonds and van der Waals forces, and this result was in accordance with that discussed above. As shown in Figure 3C, there is a hydrogen bond between the H atom at position 12 of MBI and the O atom on Gly 51 of chain A. A hydrogen bond also exists between the N atom at position 7 of MBI and the hydrogen atom HN on Val 7 of chain B. Electrostatic interactions and hydrophobic forces also existed, but hydrogen bonds and van der Waal forces played a major role in the binding of MBI to Cu/ZnSOD.
(A) Binding site of MBI to Cu/ZnSOD. Two subdomains of Cu/ZnSOD are in different colors. (B) 2D structure of MBI with atom numbers. (C) Detailed illustration of the binding between MBI and Cu/ZnSOD. Hydrogen bonds are 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).
Investigation on Cu/ZnSOD conformation changes
UV-vis absorption spectroscopy.
UV-visible absorption spectroscopy can be used to explore protein structural changes and to investigate protein-ligand complex formation. The UV-vis absorption spectra of Cu/ZnSOD with various amounts of MBI are shown in Figure 4. The strong absorption peak at approximately 202 nm reflects the framework conformation of the protein [30]. When the concentraion of MBI increased, the absorbance of Cu/ZnSOD decreased, and the maximum peak position of MBI-Cu/ZnSOD was red-shifted by 2 nm. The results indicated that the interaction between MBI and Cu/ZnSOD led to the loosening and unfolding of the protein skeleton [40].
Conditions: Cu/ZnSOD: 1.0×10−6 molL−1; MBI/(×10−5 molL−1): (a) 0, (b) 1, (c) 2, (d) 3; pH 7.4; T = 293 K.
Synchronous fluorescence.
Synchronous fluorescence spectroscopy can give information about the molecular microenvironment in the vicinity of chromophores such as tryptophan and tyrosine because the shifts in the emission maximum are related to the changes in the polarity of their environment. When the wavelength interval (Δλ) is fixed at 15 or 60 nm, the synchronous fluorescence gives characteristic information of tyrosine residues or tryptophan residues, respectively [41].
With the increasing concentration of MBI, the emission maximum of tyrosine residues (in Figure 5A) and tryptophan residues (in Figure 5B) had blue shifts, demonstrating that the conformation of Cu/ZnSOD was changed such that the polarity around the tyrosine and tryptophan residues decreased and the hydrophobicity increased. It is shown in Figure 6 that the slope was higher when Δλ was 15 nm revealing that MBI was closer to the tyrosine residues than to the tryptophan residues. Because the binding site of MBI was closer to the tyrosine residues, the microenvironments of the tyrosine residues were influenced more than those of the tryptophan residues.
Conditions: Cu/ZnSOD: 3.0×10−6 molL−1; MBI/(×10−5 molL−1): (a) 0, (b) 1, (c) 2, (d) 3, (e) 4, (f) 5; pH 7.4; T = 293 K.
Conditions: Cu/ZnSOD: 3.0×10−6 molL−1; (○)Δλ = 15 nm and (▪) Δλ = 60 nm.
Circular dichroism.
To further ascertain the possible influence of MBI on the secondary structure of Cu/ZnSOD, CD spectroscopy was performed. The CD spectra of Cu/ZnSOD treated by various concentration of MBI were detected in Figure 7. We have performed the analysis of CD spectra by using the CDPro software package and summarized the results from the CD analysis for four secondary structures: α-helix, β-sheet, turns and unordered (Table 4) [42]. Cu/ZnSOD has the secondary structures of 8.3% α-helix, 39.0% β-sheet, 21.8% turns and 31.1% unordered. With the addition of MBI to Cu/ZnSOD (1∶20), the α-helix decreased by 0.5%, the β-sheet increased by 0.7%, turns increased 0.4% and unordered section decreased by 0.7%. The results indicated that the binding of MBI with Cu/ZnSOD induced some secondary structure changes in Cu/ZnSOD. On the basis of the above experimental results, the binding of MBI to Cu/ZnSOD induced conformational changes in Cu/ZnSOD and MBI had an obvious denaturing effect on Cu/ZnSOD.
Conditions: Cu/ZnSOD: 5.0×10−7 molL−1; pH 7.4; T = 293 K.
Effect of MBI on Cu/ZnSOD activity
The structure of a protein is related to the function such that a structural variation may affect its normal physiological function. It can be seen from the above data that the effect of MBI on Cu/ZnSOD conformation is obvious. Therefore, the effects of different concentrations of MBI on the activity of Cu/ZnSOD in vitro at physiological pH 7.4 were investigated (Figure 8). The relative Cu/ZnSOD activities were reduced to 97.5±1.2 and 93.7±1.2% after treatment with 30 and 100 nM MBI for 60 min. As the exposure time increased to 240 min, the activities were reduced to 94.7±1.5 and 89.3±1.2%. The results indicated that the MBI concentration had a significant effect on Cu/ZnSOD activities. However, the incubation time had little effect on the enzyme activities. Based on the docking result, MBI did not directly bind into the Cu/ZnSOD activity site, but the binding of MBI into the enzyme interface of two subdomains influenced the microenvironments of the Cu/ZnSOD activity sites which resulted in the reduced Cu/ZnSOD activity.
Cu/ZnSOD was exposed to graded concentrations of MBI for 60, 120 or 240 min. Conditions: Cu/ZnSOD: 3 nM. Data were pooled from at least three independent experiments and analyzed with one-way ANOVA. Error bars indicate SD.
Conclusions
In this paper, the toxic interaction of MBI with Cu/ZnSOD was carried out by multiple spectroscopic and molecular docking methods under simulated physiological conditions. MBI can spontaneously bind with Cu/ZnSOD to form a MBI-Cu/ZnSOD complex with one binding site through hydrogen bonds and van der Waals forces. The UV-visible absorption, synchronous fluorescence and CD spectra indicated that the microenvironment and secondary structure of Cu/ZnSOD were altered in presence of MBI. MBI bound into the Cu/ZnSOD interface of two subdomains. Because the binding of MBI affected the microenvironment of the Cu/ZnSOD activity site, MBI led to the inhibition of Cu/ZnSOD activity. This work provides important insights into the interaction mechanism of MBI with Cu/ZnSOD.
Supporting Information
Figure S1.
Plot of lg[(F0–F)/F] versus lg[Q] for the interaction of MBI and Cu/ZnSOD at 293 and 310 K.
https://doi.org/10.1371/journal.pone.0106003.s001
(DOC)
Author Contributions
Conceived and designed the experiments: YT LZ. Performed the experiments: YT MH. Analyzed the data: YT. Contributed reagents/materials/analysis tools: YT MH YC. Contributed to the writing of the manuscript: YT. Corrected the paper: LZ.
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