Elsevier

Food Chemistry

Volume 309, 30 March 2020, 125743
Food Chemistry

Study on the molecular interactions of hydroxylated polycyclic aromatic hydrocarbons with catalase using multi-spectral methods combined with molecular docking

https://doi.org/10.1016/j.foodchem.2019.125743Get rights and content

Highlights

  • 1-OHNap, 9-OHPhe or 1-OHPyr can bind with catalase (CAT) to varying degrees.

  • The specific interaction modes of the three OH-PAHs with CAT differed obviously.

  • The three OH-PAHs induced varied conformational changes of CAT.

  • The activity of CAT was promoted by 9-OHPhe, but inhibited by 1-OHNap or 1-OHPyr.

Abstract

To reveal the potential effects of hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) on catalase (CAT), the interactions of 1-hydroxynaphthalene (1-OHNap), 9-hydroxyphenanthrene (9-OHPhe) and 1-hydroxypyrene (1-OHPyr) with CAT were investigated using multi-spectroscopic and molecular docking techniques. Fluorescence analysis showed that 1-OHNap, 9-OHPhe and 1-OHPyr can form 1:1 complex with CAT, with the binding constant of 6.31 × 103, 1.03 × 104 and 2.96 × 105 L mol−1 at 17 °C. Thermodynamic and docking parameters demonstrated that van der Waals’ force, hydrogen bonds and hydrophobic interactions dominated the three binding processes. Molecular docking also revealed the specific binding mode of OH-PAHs with CAT. Synchronous fluorescence and circular dichroism spectral results indicated that the three OH-PAHs induced varied structural changes of CAT. Furthermore, CAT activity was promoted by 9-OHPhe, but inhibited by either 1-OHNap or 1-OHPyr. Under the maximum experimental concentration of OH-PAHs, the percent change of CAT activity induced by 1-OHNap, 9-OHPhe and 1-OHPyr were 8.42%, 4.26% and 13.21%.

Introduction

Polycyclic aromatic hydrocarbons (PAHs), as typical persistent organic pollutants, can accumulate in human beings through food intake, as they may be formed during the process of food preparation, such as barbecuing, smoking, or frying (Gysel et al., 2018, Lao et al., 2018). After entering the human body, parent PAHs can be primarily metabolized by cytochrome P450 enzymes and converted to more active oxy-derivatives such as epoxides and hydroxyl compounds. These metabolites have the potential ability to bind with biomacromolecules (e.g., DNA and proteins), which may induce damage to the DNA or the proteins (Moorthy, Chu, & Carlin, 2015). Therefore, the investigations on the molecular interactions between biomacromolecules and the oxygenated metabolites of PAHs are of importance to understand the toxic effects of PAHs. Hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs), a type of important metabolites of PAHs, are considered to be potentially more toxic than their parent PAHs (Diamante et al., 2017), while they have not been studied extensively as their parent PAHs. Hence, more attention should be paid to the molecular interactions of OH-PAHs with functional biomacromolecules and the corresponding effects.

Catalase (CAT, EC 1.11.1.6) is a heme containing metalloenzyme, which catalyzes the degradation of hydrogen peroxide (H2O2) into one molecule of H2O and a half molecule of O2 (Deisseroth & Dounce, 1970), thus playing an important role in protecting cells from oxidative injure induced by reactive oxygen species (ROS). As is well-known, ROS have been considered to be an important factor in aging, cancer, diabetes, hypertension and inflammation, and thus the denaturation of CAT is correlated with these pathological states (Glorieux & Calderon, 2017). Recently, more and more studies have been performed on the potential toxicity of pollutants on CAT in vivo and in vitro. Intake of exogenous environmental pollutants is suggested to cause the conformational destruction of CAT and/or to affect the catalytic activity of CAT (Chen et al., 2018, Wang et al., 2016, Xu et al., 2018). Even so, the toxicity mechanism of pollutants on CAT is still far from being fully understood.

As the toxicity of contaminants is originated from their interactions with functional biomacromolecules (Moorthy et al., 2015), the investigation on the molecular interactions of OH-PAHs with CAT are crucial to reveal the toxicity mechanism of OH-PAHs on the antioxidant enzyme system. In our previous work, the interaction between CAT and 1-hydroxypyrene (1-OHPyr) were preliminary investigated (Chen, Zhang, Zhu, & Zhang, 2015), and the strong binding affinity of 1-OHPyr to CAT and the adverse effect of 1-OHPyr on the conformation and function of CAT were confirmed. However, minor changes in the structure of OH-PAHs may induce their different interaction behaviors with biomacromolecules and govern their toxicity in organisms (Sievers et al., 2013, Wang et al., 2009). Wu et al. (Wu, Wu, Guan, Su, & Cai, 2007) proved the formation of the complex between hydroxynaphthalene and bovine serum albumin (BSA), and revealed that the binding of 1-hydroxynaphthalene (1-OHNap) with BSA was more cooperative than that of 2-hydroxynaphthalene. Wang et al. (2009) investigated the binding of 26 OH-PAHs with DNA, which suggested that steric effect was vital in the interaction of DNA with differently substituted OH-PAH. Ohura, Kurihara, and Hashimoto (2010) investigated the binding interactions between OH-PAHs and aryl hydrocarbon receptor (AhR), and the results indicated that the number and site of hydroxy-groups substituted on PAH skeleton obviously influenced the AhR-ligand binding capacity of OH-PAHs, with the octanol-water partition coefficient (Kow) value of OH-PAH significant related to the AhR activity. Li et al. (2011) developed a QSAR model to characterize the interactions of OH-PAHs with DNA, which suggested that the electrostatic potential, molecular size and polarizability of OH-PAHs were closely correlated with their binding capacity to DNA. Sievers et al. (2013) reported that 1-OHNap, 9-hydroxyphenanthrene (9-OHPhe) and 1-hydroxypyrene (1-OHPyr) were able to competitively bind ERβ, induce ERβ homodimers, and regulate ERβ target genes, whereas, the relative activity of each compound is quite different. These studies confirmed that the binding affinity/the adverse effects of OH-PAHs to/on biomacromolecules varied as the structural difference of OH-PAHs. Specifically, the logKow, charges and steric effect of OH-PAHs are important factors affecting the binding interactions. Therefore, in order to fully understand the effects of the structural difference of OH-PAHs on their interactions with CAT, further work on different OH-PAHs with varied number of benzene rings should be conducted.

Herein, based on our previous work, the interactions of CAT with 2–4 ring monohydroxy PAHs (1-OHNap, 9-OHPhe and 1-OHPyr) were further investigated in the present study. Briefly, this work is undertaken based on the following hypothesis: 1) OH-PAHs with different numbers of aromatic rings may have varied binding affinity to CAT, and the binding ability of CAT with OH-PAHs may be associated with the hydrophobicity of OH-PAHs; 2) OH-PAHs with different numbers of aromatic rings can induce different structural and functional changes of CAT, due to the varied molecular interaction of OH-PAHs with CAT. To verify these hypotheses, firstly, multi-spectroscopy, in combination with molecular docking method, was employed to study the binding information of OH-PAHs with CAT. Then, synchronous fluorescence and circular dichroism (CD) spectroscopy methods were employed to clarify the effects of the binding interactions on the structure of CAT. Enzyme activity measurements were further utilized to interpret the response of CAT activity after OH-PAHs exposure. Meanwhile, the differences of the interactions of CAT with OH-PAHs with different numbers of aromatic rings were discussed throughout the study. These investigations aim to help clarify the molecular mechanism of the interactions between CAT and OH-PAHs, and provide an important basis for further understanding the toxicity of OH-PAHs on antioxidant enzyme systems in vivo and further warn the health risk of the PAHs via dietary intake.

Section snippets

Materials

Three OH-PAHs (1-OHNap, 9-OHPhe, and 1-OHPyr) with purity > 99% and CAT from bovine liver were purchased from Sigma-Aldrich Company, USA. 30% H2O2 was from Xilong Chemical Company, Ltd, China. Stock solutions of 5.0 × 10−5 mol L−1 CAT were prepared in Tris-HCl buffer. 1.0 × 10−2 mol L−1 1-OHNap, 2.0 × 10−3 mol L−1 9-OHPhe and 2.0 × 10−3 mol L−11-OHPyr were all prepared in ethanol and stored at 4 °C in the dark. All chemicals were of analytical grade, and ultrapure water (>18.2 MΩ cm−1) was used

Effects of 1-OHNap, 9-OHPhe or 1-OHPyr on the fluorescence spectra of CAT

The intrinsic fluorescence of CAT is mainly attributed to its tryptophan (Trp) and tyrosine (Tyr) residues, and there are 6 Trp and 20 Tyr residues in each subunit of CAT (Schroeder et al., 1982). The fluorescence spectra of CAT with various concentrations of 1-OHNap, 9-OHPhe or 1-OHPyr were shown in Fig. 1. As shown in Fig. 1, pure CAT displays a strong fluorescence emission peak at 350 nm upon excitation at 280 nm. However, the fluorescence emission intensity of 1-OHNap, 9-OHPhe and 1-OHPyr

Conclusion

In this study, the interactions of three OH-PAHs with CAT and the corresponding effects on the structure and function of CAT at the molecular level were investigated using experimental studies and molecular docking. The results showed that 1) the binding affinity of the three OH-PAHs with CAT increased with the increment of the number of benzene rings at 17 and 27 °C, as the hydrophobicity of OH-PAHs could affect their binding ability to CAT. However, the order of the binding constants at 35 °C

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We gratefully acknowledge the financial support of the National Major Scientific Instruments Development Project of China [grant number 21627814], the National Natural Science Foundation of China [grant number 21577110], the Natural Science Foundation of Fujian Province [grant number 2018J05024] and the Program for Prominent Young Talents in Fujian Province University, China [grant number [2018]47].

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