Electrochemistry and molecular modeling of the hemoglobin–benzene interaction with a nanocrystalline mixed metal oxide

Abstract

Nanocrystalline mixed metal oxides of various metal cations were synthesized and were then used for the construction of a new modified carbon paste electrode. Hemoglobin (Hb) was entrapped on the surface of this electrode using an alginate biocompatible biopolymer that crosslinked with the calcium ions. Spectroscopic studies were done to determine the characteristics of the biopolymer matrix and the effects of benzene on Hb. The direct electrochemistry of Hb using the proposed method and its application in Hb interaction with benzene were investigated using an electrochemical method. The heme complexes with imidazole and histidine were also entrapped on the surface of the electrode and the effect of benzene on these complexes was also studied. The Hill binding parameters were obtained for the Hb–benzene, imidazole-heme–benzene and histidine-heme–benzene interactions. A molecular docking simulation was used for theoretical studies and the data obtained were compared with experimental results. A good agreement was observed between the theoretical and experimental results. The Hb electrode can be used for benzene determination in water and aqueous solutions because of the linear relationship between benzene concentration and the peak current decreases with good linearity. The proposed sensor has advantages such as it has an easy, simple and inexpensive preparation method, shows good linearity and low detection and good quantification limits, good reproducibility and sensitivity, and has a relatively long life time (about 15 days) that is because of the alginate biopolymer biocompatible matrix.

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1. Introduction

Electron transfer has a fundamental role in many biological phenomena such as respiration, photosynthesis, nitrogen fixation, enzyme functions, immune response, and so on.¹ Direct electron transfer between the redox macromolecules and the surface of electrodes can be used to investigate the enzyme-catalyzed reactions in biological systems and to create the electrochemical basis for the study of the structure of redox proteins, kinetics and thermodynamics of redox transformations of electroactive macromolecules, and metabolic processes involving redox transformations.^2–6 Protein electrochemistry has gained more interest in recent years because of its potential applications in biosensors, bio-fuel cells and bioreactors.^5,7–13 The mechanism of direct electron transfer between macromolecules and the electrode surface can also provide useful information on the mechanism of bioreactions, structure variation, ligand binding and the electron transfer process in biological systems.^14–16 Because of the deep burying of the electroactive prosthetic groups, the partly adsorptive denaturation of the proteins on the electrode surface or the unfavorable orientations at the electrodes, the heterogeneous electron transfer process of bio-macromolecules is rather slower at the traditional electrode surface in comparison with native proteins. The aim of research in this field is to focus on establishing different kinds of promoters or the supporting films to facilitate the direct electron transfer between the electroactive prosthetic group and the electrode surface. Different kinds of immobilization methods have been successfully used for construction of protein modified electrodes.^4,14–20 The encapsulation and immobilization methods make a suitable chemical microenvironment for the proteins to keep their native structures and functions because this gives the protein molecules more freedom in orientation. Under these conditions, direct electron transfer between the redox macromolecule and the electrode surface can be easily achieved. Various methods and compounds have been used for this purpose such as surfactants, hydrogel polymers, nanoparticles, biomembranes and polyelectrolyte layer-by-layer films.^14–18,21 Biopolymers such as alginate (Alg), which is a versatile biopolymer, have been widely used as an immobilization matrix for biosensors and biocatalysis because of their good biocompatibility, non-toxicity, biodegradability and excellent film forming ability.^15,19,22

Hemoglobin (Hb) is an iron containing heterotetramer protein that is carried by red cells. Each red blood cell contains approximately 280 million Hb molecules. Hb in the blood carries oxygen from the lungs to the tissues and then transports carbon dioxide back from the organs to the lungs and also regulates the pH of the blood.²³ Hb exists in two forms: the taut form (T) and a relaxed form (R). Hb functions by making structural changes to itself and the R state is common in lungs and the T state is common in the capillaries. A reduction in the total binding capacity of Hb to oxygen because of a reduced pH is called the root effect and as a result of this effect, Hb has cooperative interactions at a high pH (in lungs and the R state) and shows non-cooperative interactions at a low pH (in capillaries and the T state).²⁴ Many factors can impact on the normal physiological functions of Hb. This macromolecule can be reversibly affected by many kinds of endogenous physiological conditions (pH, temperature, and so on), and exogenous agents and environmental pollutants (drugs, heavy metals, herbicides (such as Alachlor, Aminopielik D and Paraquat), and insecticides (such as Chlorpyrifos and Cypermethrin) and organic pollutants, and so on) that can alter protein structure and function.²⁵ Hb consists of two α-subunits and two β-subunits. Each subunit contains a prosthetic heme group that cooperatively binds and releases oxygen so that each Hb molecule has the ability to transport up to four oxygen molecules. The heme prosthetic group (iron porphyrin) in each subunit acts as the electroactive center. The electron transfer between a heme protein (e.g., myoglobin, Hb) in solution and the bare electrode is either not observed or is very slow.²⁶ Hb is involved in various blood dyscrasias and many clinical diseases such as non-immune hemolytic anemia, leukemia, methemoglobinemia, heart disease, excessive loss of blood and so on.^5,25,27–29

Benzene, toluene, ethylbenzene, and xylenes (BTEX compounds) are some of the volatile organic compounds (VOCs) found in petroleum derivatives such gasoline or in several organic and industrial solvents.³⁰ These chemicals are classified as hazardous air pollutants (HAPs).³¹ Exposure to HAPs can cause a variety of health problems such as cancer, respiratory irritation, and central nervous system damage. Toluene, ethylbenzene, and xylenes have a harmful effect on the central nervous system. Based upon toxicity data, benzene represents the greatest threat of the BTEX compounds present in landfill gas, because it is a known carcinogen.^32–34 Benzene increases the risk of various illnesses such as aplastic anemia, acute leukemia, bone marrow abnormalities and failure. Benzene is associated with certain hematologic malignancies such as acute myeloid leukemia, myleodysplastic syndrome, acute lymphoblastic leukemia, and chronic myeloid leukemia. The department of health and human services in the United States (US-DHHS) placed benzene in group A of human carcinogens.^29,35,36 In 1948, the American Petroleum Institute (API) stated that “the only absolutely safe concentration for benzene is zero”. The affinity that benzene has for various macromolecules in both the water-soluble and water-insoluble states is of particular interest because benzene has the prototypic physical chemical properties of many VOCs. Certain previously reported aromatic compounds can make complexes with the heme moiety of the myoglobin that results in conformational changes in the protein. Benzene and chlorpromazine were found to have a large and specific enhancing effect on the rate of reaction of zinc ions with myoglobin and on the rate of denaturation of this protein by urea. Because of the fact that the effect of aromatic compounds on the denaturation of apomyoglobin is very small, the importance of the heme moiety of the molecule in binding with benzene compounds is clear.³⁷ These results indicated that aromatic compounds interact with myoglobin at both the globin and heme parts of myoglobin. The effect of hydrophobicity of aromatic compounds in the interaction with heme proteins and its penetration to hydrophobic pocket is a hard question for researchers and needs further study. For these studies we need powerful techniques that can validate the hydrophobic effects and interactions in the heme moiety and in the globin of heme proteins.^37,38 Spectroscopic methods are not good for this purpose because aromatic compounds interfere in spectrophotometric studies and the volatility of these compounds limit the experimental work that can be done. High sensitivity, fast analysis time, repeatability, simplicity, availability, inexpensive materials and methods are all crucial in these studies. Electrochemical studies have various advantages and because of this fact they are of great interest for chemical and biological research. Research on the molecular mechanisms of toxicity of environmental contaminants and methods to evaluate the toxicity of aromatic organic compounds are at the forefront of environmental toxicology today.^34,38 In this research, we focused on the synthesis and application of nanocrystalline mixed metal oxide (MMO) for modifying a carbon paste electrode (CPE) and subsequent application of alginate for immobilization of Hb, heme-imidazole and heme-histidine electroactive compounds on the electrode surface that subsequently crosslinked with sodium polyphosphate or calcium ions for effective immobilization, respectively. The proposed electrode is biocompatible, and uses easy, fast, reliable and inexpensive immobilization and electron transfer between Hb and the synthesized nanoparticles. This is a novel application of electrochemistry to the binding studies of benzene as a volatile aromatic hydrocarbon with Hb and macromolecules. Besides accurate binding studies, the proposed electrode was used for benzene determination. Direct electrochemistry of Hb and the effect of benzene on the electrochemical behavior of Hb can help to understand the heme degradation mechanism and the pulling out of the heme from the hydrophobic pocket by comparing the electrochemical behavior of imidazole or histidine complexes of the heme prosthetic group.

2. Experimental

2.1. Materials

Alginic acid was obtained from Sigma Chemical Company. All the other reagents and chemicals were purchased from Merck and were of analytical grade and used without further purification. All the solutions were prepared with doubly distilled water.

2.2. Human adult hemoglobin extraction

Human hemoglobin (hHb) was extracted in our laboratory using a method described previously.²³ New, fresh and heparinized blood was centrifuged at 3000 rpm to remove plasma components. The upper yellowish solution was decanted and the packed red cells were washed three times in an isotonic saline solution (0.9% NaCl) at a ratio of 1 : 10, for 5 min and subsequently centrifuged three times at 10 000 rpm. Red blood cells were osmotically lysed with cold double distilled water. Membrane components were removed by centrifugation (10 000 rpm). The soluble Hb was centrifuged at least twice more at high speed to remove any insoluble materials (18 000 rpm). The Hb solution was then brought to 20% saturation with ammonium sulfate, left to stand for about 15 min, and then centrifuged at 20 000 rpm. The resulting Hb solution was dialyzed three times for 24 h against 0.2 M phosphate buffer solution (PBS; pH 7.4).

2.3. Molecular docking analysis

MGL Tools v1.5.4 with Autogrid 4 and Autodock 4 were used to obtain the binding energy, and the binding sites between benzene with human hemoglobin (hHb).³⁹ The crystal structure of oxyhemoglobin was obtained from the protein data bank (PDB ID:1HHO). Receptor (hHb) and ligand (benzene) files were prepared using Autodock Tools. The hHb was enclosed in a box with number of grid points in the x × y × z directions, 126 × 126 × 126 and a grid spacing of 0.458 Å. A Lamarckian genetic algorithm was applied in the docking and 100 genetic algorithm runs were performed. For each of the docking cases, the lowest energy docked conformation, according to the Autodock scoring function, was selected as the binding mode. The most favorable docked structure of the hHb–benzene complex was viewed and handled with PyMol software.⁴⁰

2.4. Synthesis of nanocrystalline mixed metal oxide

The Cu–Zr–Ce mixed oxide nanocrystalline particles were produced by dropwise addition of an aqueous solution of sodium hydroxide into a stirred, mixed aqueous solution of Ce, Cu and Zr solutions (by considering the appropriate amounts of each cation) in a beaker until the pH of the solution was 10 using procedures reported previously.⁴¹ The resulting precipitate was left stirring for 6 h for further ageing of precipitate. After completion of the precipitation process, the sediment was washed several times and then dried at 120 °C overnight, followed by calcination at 850 °C for 6 h in an air atmosphere. The field emission scanning electron microscopy (FE-SEM) images for the nanocrystalline Ce–Cu–Zr synthesized are presented in Fig. 1.

Fig. 1 FE-SEM image for the nanocrystalline Ce–Cu–Zr MMO synthesized.

2.5. Apparatus

Electrochemical studies were carried out in a conventional three-electrode cell powered with a computerized potentiostat/galvanostat (PalmSens, Netherlands). An Ag/AgCl-saturated KCl electrode, a Pt electrode (Azar Electrode, Iran) and a nanoparticle modified carbon paste (NCP) electrode were used as the reference, counter and working electrodes, respectively. FE-SEM (Hitachi S 4160, Japan) was used for nanoparticle characterization.

2.6. Electrode preparation

For the NCP electrode preparation, 0.05 g of the precipitate obtained (nanocrystalline particles of Cu–Zr–Ce oxide) was mixed with 0.5 g graphite (1 : 10). Then two drops of paraffin oil were added to the mixture. The mixture was housed and packed in a polyethylene syringe (inner diameter 2.5 mm) and its tip was polished on a smooth paper layer. Also an electric contact was made by copper wire through the back of the electrode.

Alginate biopolymer was used for Hb entrapment on the surface of the NCP electrode. For this purpose the concentration ratio of biopolymers-binder-Hb was optimized. To PBS (pH 7.4) containing alginate (2 mg mL⁻¹) an appropriate amount of Hb was added and the solution was mixed well until a homogeneous solution was obtained. A drop of the prepared mixture was placed on the surface of an NCP electrode and immersed in a suitable binder solution (calcium solutions as a crosslinking agent for alginate).

The modified electrode was rinsed with double distilled water and then inserted in an electrochemical cell containing PBS (2 mM, pH 7.4). The Hb cyclic voltammograms (CVs) were recorded frequently until a stable electrochemical response was observed. The prepared modified electrode was stored at 4 °C when not in use. It was stable for at least two weeks.

The other modified electrodes were also prepared using heme-imidazole or heme-histidine complexes, following a similar procedure to that used for Hb. The electrochemical measurements were carried out in a 25 mL electrochemical cell containing 2 mM PBS at pH 7.4. For removing the dissolved oxygen, before a series of experiments the sample solutions were purged with highly purified nitrogen for 30 min.

3. Results and discussion

3.1. Spectroscopy

The ultraviolet-visible (UV-Vis) spectra of Hb (particularly in the Soret band, B band and Q band regions) may provide information about the possible denaturation of heme proteins, and particularly on the conformational change in the heme group region. Therefore, UV-Vis spectroscopy is a useful tool for the conformational study of the heme region. In Hb denaturation, the Soret band may disappear partly or completely. The UV-Vis absorption spectra of Hb in the presence of alginate in PBS (pH 7.4) are similar to native Hb. However, a small hyperchromicity can be seen in the Soret peak because of the increase in hydrophobicity caused by the biopolymeric matrix. Because the change in the Soret band was negligible, it is clear that Hb in the presence of a biopolymer film was not distinctly denatured. The absorption spectrophotometric spectra of Hb in the presence of benzene was also recorded to determine the structural and functional effects of ligand on the Hb. The Hb spectra in the absence and presence of benzene was recorded. By increasing the benzene concentration, hypochromic and any bathochromic shifts in the Soret band region can be seen. Also, a hypochromic decrease was observed in the Q bands (Fig. 2). Q bands are related to the oxy and de-oxy structure of Hb and revealed the oxygen carrying function of this macromolecule. Benzene interaction with Hb was studied in the oxy and deoxy forms of a macromolecule to help understand the effect of the oxy and deoxy forms of Hb on benzene interaction. The results demonstrated that the oxygen bonded form of Hb showed a low potential for benzene interaction at the heme center. By removing the oxygen from the experimental solution, the affinity of Hb to benzene was increased. Our studies on the oxygen affinity of Hb in the presence of benzene demonstrated that benzene extensively affected the oxygen affinity of Hb. The oxygen was removed from Hb solution by purging it with N₂. Then the benzene was added to the solution and the oxygen affinity of the Hb was measured. The sigmoid shape of the affinity plot was changed in the presence of benzene and the plot shifted to the left which proved that the hydrophobicity around the heme was increasing (data not shown). Decomposition of the affinity plot to an irregular shape and its left shift proves the intensive affinity of oxygen decreases the effect that benzene has on the Hb. Benzene and oxygen have a competitive effect for the oxygen binding site and the results obtained here demonstrate that after benzene binds to the site, oxygen cannot bond but benzene can be substituted with bonded oxygen at the oxygen site. This can be explained based on the cation–π interaction between the benzene π electrons and the iron center of the heme.^42,43 In order to verify the spectroscopic results, molecular docking analysis was considered and designed.

Fig. 2 UV-Vis spectra of Hb recorded with increasing benzene. (A) Oxygen equilibrated Hb: increase of benzene concentration from 1 × 10⁻⁸ to 2 × 10⁻⁶ M induced a decrease in Soret band (dashed arrow) and Q bands (both solid arrows). (B) N₂ purged Hb solution (O₂ removal): increase of benzene concentration from 1 × 10⁻⁸ to 1.5 × 10⁻⁷ M induced a larger decrease of the Soret band (dashed arrow) and Q bands (both solid arrows) in comparison with (A), at room temperature and pH = 7.4.

3.2. Molecular docking

In order to further locate the binding sites of benzene on hHb, the complementary application of computational modeling has been employed using Autodock 4.0. The molecular docking technique is an attractive method for determining the ligand–protein interactions which can help to prove the experimental results. Also, the docking programs, when used with experimental screening, can help in better understanding of bioactivity mechanisms and functions related to the target macromolecule. The best simulation results are shown in Fig. 3. As can be seen, benzene is situated in the hydrophobic pocket of hHb near to the heme center of protein.

Fig. 3 Minimum energy docking conformation (obtained from molecular docking simulation). The hHb and benzene are represented by ribbon structure and sphere model, respectively. A closer view of benzene penetration in the hydrophobic pocket and its orientation: (A) oxygen bonded heme and (B) oxygen released heme.

Hb has a globular shape with reverse turns that contribute to the circular shape of the protein. The Hb quaternary structure comes from its four subunits which are in a roughly tetrahedral arrangement. This protein is a tetramer consisting of two identical α-subunits and two identical β-subunits. Each subunit is composed of a protein chain that is tightly associated with a prosthetic heme group. Each chain arranges itself into a set of α-helical structural segments connected together in a globin fold arrangement, so called because this arrangement is the same folding motif used in other heme proteins such as myoglobin. This folding pattern contains a pocket that strongly binds the heme group.⁴⁴

The molecular docking results are presented in Table 1 and the dominating configuration of the binding complex with minimum binding free energy (ΔG) is shown in Fig. 3. The docked structure (Fig. 3A) shows that benzene is located in a hydrophobic pocket of the Hb. Also looking more closely into the docked structure, the benzene is seen to be oriented to the oxy site of the heme group in the pocket (Fig. 3B). According to the results obtained from spectroscopic studies (Fig. 2) it is clear that in the presence of oxygen in the related binding site, benzene cannot get too close to the heme group because of the prevention effect of oxygen. These results are in agreement with experimental data. By slowly purging the sample solution of N₂, the oxygen concentration in the solution is decreased and so the bond oxygen is released from the pocket in the Hb and benzene can come close to the oxy site of the prosthetic group and affect the structure and function of the Hb such as causing hypochromic effects in the B and Q bands of the UV-Vis spectra. Because of this fact, that the hydrophobic benzene is attracted to the hydrophobic sections of Hb, we frequently see the great affinity of benzene to the hydrophobic pocket of the Hb which is in agreement with the theoretical and experimental results obtained. This proved that oxygen has a structural role and stability effect on Hb.

Table 1 Hill parameters and molecular docking data for the interaction of benzene with immobilized Hb and heme complexes

Complex (immobilized in alginate)	Hill parameters			Free energy (kcal mol^−1)
	nH	log KH	R^2	Theoretical	Experimental
Hb	1.23	5.82	0.98	−7.92	−4.30
Heme-imidazole	1.15	5.71	0.98	—	—
Heme-histidine	2.25	10.35	0.99	—	—

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3.3. Electrochemical experiments

Electrochemistry is one of the most powerful techniques in chemical and biochemical interaction studies and for understanding the molecular mechanism of reactions. In this study the electrochemical experiments were designed for detailed study and understanding of the benzene–Hb interaction and evaluation of theoretical and spectroscopic results. As mentioned in previous sections, direct electrochemistry of proteins is of great interest in biochemical research. Some problems concerning the electrochemistry of proteins in conventional methods have arisen previously such as slow electron transfer to the electrode and protein denaturation. For solving problems, new modified electrode and immobilization methods are investigated. In this research, a biopolymeric matrix for immobilizing Hb for direct electrochemistry of Hb a new modified electrode using nano-crystalline MMO was used. Optimization of the electrochemical responses from the new modified electrode and the immobilized Hb on the electrode was necessary.

3.4. Electrochemical behavior of the modified electrode

The performance of the NCP electrode was also studied by CV. The experimental results revealed that after about 20 cycles at a scan rate of 100 mV s⁻¹ the CV scans became stable. The free metal ion contamination possibly remained in the carbon paste channels or the nanoparticle lattice during the synthesis process and these were excluded by using continuous potential scanning. In this way the electrode surface could achieve an equilibrium with the buffer solution and attain an electroneutrality in potential.⁴⁵

Fig. 4 shows the CVs of different NCP electrodes in PBS (pH 7.4) at the scan rate of 100 mV s⁻¹. No redox peaks appeared on either a bare NCP (curve a) or an alginate coated NCP electrode (curve b). However, when the Hb was entrapped in the alginate film via crosslinking in calcium solution, direct electron transfer between Hb and the modified electrode occurred and a pair of well-defined quasi-reversible redox peaks were detected (curve c), which is attributed to the heme Fe(III)/Fe(II) redox couple.

Fig. 4 CVs of (a) bare MMO-CPE, (b) Alg-Ca/MMO-CPE, (c) Hb/Alg-Ca/MMO-CPE in PBS (0.1 M, pH 7.4) at the scan rate of 50 mV s⁻¹.

The presence of Cu–Zr–Ce oxide nanocrystalline particles in the paste electrode provided a large surface area for alginate biopolymer absorption and then Hb entrapment on the surface of the NCP electrode. It also facilitates the Hb electron transfer rate. The alginate film on the electrode provided a biocompatible microenvironment to maintain the natural conformation and bioactivity of Hb.

As seen in Fig. 4, the anodic (E_pa) and cathodic (E_pc) peak potentials were obtained at 0.049 and −0.315 V vs. Ag/AgCl, respectively. The apparent formal potential (E⁰′ = E_pa + E_pc/2), was calculated to be −0.183 V vs. Ag/AgCl. The peak-to-peak potential separation (ΔE_p = E_pc − E_pa) was calculated to be 0.365 V at 100 mV s⁻¹ and the ratio of redox peak current (I_pa/I_pc) was approximately 1.08. These data obviously indicated that the direct electron transfer of Hb at alginate film on a modified electrode could be achieved.

The CVs of a modified electrode in the presence of 0.2 M PBS (pH 7.4) containing no deliberately added electroactive material were recorded between −0.7 and 0.7 V vs. Ag/AgCl at various scan rates from 50–250 mV s⁻¹.

By increasing the scan rate, the peak separation also increased. This indicated a limitation arising from charge transfer kinetics (Fig. 5). The peak currents of the CVs were linearly proportional to the scan rate up to 250 mV s⁻¹, which is expected for surface confined redox process (Inset A, Fig. 5). E_p was proportional to ln(v) (Inset B, Fig. 5).

Fig. 5 CVs of an Hb-modified electrode at various scan rates (50, 100, 150, 200, 250 mV s⁻¹) (increasing of potential scan rate shown with arrows). Inset A: redox peak current (I_p) versus scan rate (ν). Inset B: the peak potential against log ν.

3.5. The interaction of Hb with benzene

The prepared electrode was used to investigate the interaction of benzene with Hb. Fig. 6 shows a series of CVs obtained by the modified electrode in PBS (0.2 M, pH 7.4) containing various amounts of benzene. It was observed that the cathodic and anodic peak current of Hb decreased gradually with addition of different concentrations of benzene. The Hill coefficient (n_H) which indicated that benzene was cooperatively bound to Hb was calculated, using eqn (1),⁴⁶ to be 1.23 (Inset, Fig. 6). The logarithm of the binding constant was 5.81, suggesting that the interaction force between them was of the intermolecular weak interaction type.where ΔI is the difference of the peak current obtained in the absence and presence of benzene and ΔI_max is the maximum of the difference of the peak current.

Fig. 6 Typical CVs of Hb immobilized on a modified electrode at various concentrations of benzene (from outer to inner: 5 to 55 μM (decrease in peak currents shown with arrow)) at the scan rate of 150 mV s⁻¹, room temperature and pH 7.4. Upper left-hand inset, calibration curve for benzene determination using the proposed modified electrode. Lower right-hand inset, logarithmic Hill plot of Hb in the presence of different concentrations of benzene (data were calculated using eqn (1)).

Further studies were carried out by replacing Hb with either heme-imidazole or heme-histidine complexes. The prepared electrode was used for evaluation of the interaction between benzene and the complexes. Typical CVs for the interaction of heme-histidine complex with benzene are shown in Fig. 7. Using eqn (1), related binding coefficients and constants (Table 1) could be obtained. The results obtained for the interaction of benzene with heme-histidine and heme-imidazole complexes are presented in the insets of Fig. 7 and 8, respectively. The corresponding Hill parameters are summarized in Table 1. All of the interactions are positively cooperative (n_H > 1). The cooperativity of benzene in interaction with imidazole-heme, Hb and histidine-heme all increased.

Fig. 7 Typical CVs of heme/histidine complex immobilized on a modified electrode at various concentrations of benzene (from outer to inner: 5 to 55 μM (decrease in peak currents shown with arrow)) at the scan rate of 150 mV s⁻¹, room temperature and pH 7.4. Inset shows the logarithmic Hill plot for benzene interaction with the heme-histidine complex entrapped on the modified electrode (data were calculated using eqn (1)).

Fig. 8 Typical CVs of the heme-imidazole complex immobilized on a modified electrode at various concentrations of benzene (from outer to inner: 5 to 55 μM (decrease in peak currents shown with arrow)) at the scan rate of 150 mV s⁻¹, room temperature and pH 7.4. Inset shows the Hill logarithmic plot for the benzene interaction with the heme-imidazole complex entrapped on the modified electrode (data were calculated using eqn (1)).

The imidazole-heme interaction with benzene is because of the interaction of the benzene π-electron ring with the imidazole-heme π–π interactions and hydrophobic sites on the complex. Because of the similarity between the histidine head group with imidazole, the importance of the hydrophobic tail of histidine in this interaction is manifested. Subsequently it is clear that the hydrophobic sites have a major effect on the benzene binding with the complexes. Therefore, the heme-histidine complex which is located in the Hb hydrophobic pocket is not easily available for benzene. Therefore, it is necessary for benzene to diffuse in to the hydrophobic protein pocket. However, benzene is a small molecule with a high ability for diffusing into the pocket section of Hb. By considering these facts it can be concluded that the effective interaction of benzene with Hb focused on the hydrophobic pocket of Hb. It must be mentioned that by removing the oxygen in the electrochemical experiments and consequently increasing the concentration of benzene in the reaction cell, the electrochemical peak corresponding to the Hb was immediately completely removed. This result is in agreement with the spectroscopic and theoretical results obtained that prove the affinity of benzene to the oxygen binding site on the heme group of the Hb. The electrochemical behavior of Hb is related to the redox reactions of the iron center of the heme group. A decrease in peak current when the benzene is increased shows that the iron center is blocked with benzene. In other words, as mentioned previously, benzene interacts with the iron cation because of the cation–π interaction. These interactions are non-covalent molecular interactions between a monopole adjacent cation (iron) and a quadrupole (benzene π system). Cation–π interaction energies are the same as hydrogen bonds and salt bridges. The diffused benzene molecules in the hydrophobic pocket of Hb can make a cation–π interaction with the iron cation in the heme group that results in decreasing peak currents. In methemoglobin these interactions are stronger because of the oxidation of Fe²⁺ to Fe³⁺.

From the docking simulation the free energy change of binding (ΔG) obtained for the benzene–Hb interaction was calculated to be (−4.30 kcal mol⁻¹) which is more than the experimental free energy of binding (−7.92 kcal mol⁻¹) obtained from the electrochemical data. This apparent mismatch in the free energy changes could be because of the solvent and bulk effects on the interactions. On the other hand, in the presence of a polar aqueous bulk solution, hydrophobic interactions show a higher potential and are more powerful in comparison with a non-polar solventless theoretical system. The immobilized Hb peak currents show linear dependence to the total concentration of benzene added to the sample solution (Fig. 5). The calibration curve was linear in a benzene concentration range from 5 to 55 μM (y = −104.06x + 8.98, R² = 0.985).

4. Conclusion

This study introduced a novel Ce–Cu–Zr MMO nanoparticle modified CPE and subsequent immobilization of Hb using alginate crosslinked with calcium, as a biocompatible biopolymer on the surface of the modified electrode, for direct electrochemistry of Hb. The modified electrode was used for studying benzene interaction with hHb. By considering the prosthetic group in the structure of Hb (as a heme protein) two types of interactions can be induced with benzene. Studying the benzene interaction with imidazole-heme and histidine-heme complexes as similar complexes to the prosthetic group (as electroactive center of Hb) of Hb can give information in regard to interactions with Hb. The binding data were accurately obtained using the modified electrode and the Hill parameters were estimated and reported electrochemically. The results obtained demonstrated that benzene interacts with Hb in both globin and hydrophobic pocket regions of Hb. The proposed electrode can be used as a new electrochemical sensor for benzene determination in aqueous solutions. The sensor has some advantages such as it has a fast, easy, simple and inexpensive preparation method, and it shows good linearity and low detection and quantification limits, good reproducibility and sensitivity, and has a relatively long life time (about 15 days in the refrigerator) that is because of the alginate biopolymer biocompatible matrix.

Acknowledgements

This work was supported by University of Tehran, Iran National Science Foundation (INSF), Center of Excellence in Biothermodynamics (CEBiotherm), University of Tehran and Iran National Elites Foundation (INEF).

Notes and references

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