Studies on the interaction of apigenin with calf thymus DNA by spectroscopic methods

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Highlights

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    Apigenin could insert into DNA base pairs to form a binary complex at physiological pH.

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    The acting force between apigenin and DNA mainly included hydrophobic interactions.

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    The stabilization of the ctDNA helix was increased in the presence of apigenin.

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    The increase of ion strength has no significant effect to the binding of apigenin with DNA.

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    The interaction between apigenin and ctDNA might occur via intercalative mode.

Abstract

The interaction between apigenin and calf thymus deoxyribonucleic acid (ctDNA) in a pH 7.4 Tris–HCl buffer solution was investigated by UV–Vis spectroscopy, fluorescence spectroscopy, DNA melting techniques, and viscosity measurements. It was found that apigenin molecules could intercalate into the base pairs of DNA, forming a apigenin–DNA complex with a binding constant of K310K = 6.4 × 104 L mol−1. The thermodynamic parameters enthalpy change (ΔH), entropy change (ΔS) and Gibbs free energy (ΔG) were calculated to be 7.36 × 104 J mol−1, 329 J K−1 mol−1 and −2.84 × 104 J mol−1 at 310 K, respectively. Hydrophobic interaction was the predominant intermolecular force in stabilizing the apigenin–DNA complex. Thermal denaturation study suggested that the stabilization of the ctDNA helix was increased when the apigenin binding to ctDNA as indicated by the increase in thermal denaturation temperature of ctDNA at around 5.0 °C in the presence of apigenin. Spectroscopic techniques together with melting techniques and viscosity determination provided evidences of intercalation mode of binding for the interaction between apigenin and ctDNA.

Introduction

DNA is quite often the main cellular target for study with small molecules of biological importance such as carcinogens, steroids, and several classes of drugs [1]. In recent years, many researches have been focused on interaction of small molecules with DNA. Small molecules can interact with DNA through the following three non-covalent modes: (i) electrostatic interaction, electrostatic attractions with the anionic sugar–phosphate backbone of DNA; (ii) groove binding, interactions with the DNA groove; and (iii) intercalation between the base pairs [2]. Among the three modes, the most effective mode of the drugs targeted to DNA is intercalative binding, which is related to the antitumor activity of the compound. The activity of the compound is mainly dependent on the method and intensity of interaction between DNA and compound. At present, studying the nature and dynamics for binding drug molecules to biomacromolecules (like DNA) is an active research area, which can lead to rational design and construction of new and more efficient drugs targeted to DNA [3].

Apigenin (Api., 4′,5,7-trihy-droxyflavone, Fig. 1) is a naturally occurring polyphenolic compound present in a variety of fruits, vegetables, and seeds [4], and it has a variety of physiological properties such as antioxidant, anti-inflammatory, and anticancer [5], [6], [7]. Recent study have proved that apigenin has the potential for use as an antiadipogenic agent to lower body-fat content and body-weight gain [8]. However, as far as our knowledge there is no report about detailed interaction mechanism of ferulic acid with DNA yet.

Small molecules binded with DNA generally exhibit marked changes in absorbance and fluorescence properties compared to when they are free in solution. In this work, the interaction between apigenin and DNA was investigated systematically using UV–Vis and fluorescence spectroscopic techniques together with the determination of series of thermodynamic parameters and binding constants, as well as DNA melting techniques and viscosity measurements. Based on the results it was suggested that the binding mode between apigenin and DNA might be intercalation.

Section snippets

Chemicals and reagents

ctDNA was purchased from Sigma biological Co. and used as received, which was used without further purification and dissolved in doubly distilled water at concentration of 1.0 × 10−3 mol L−1 (as stock solutions). The purity of ctDNA was checked by monitoring the ratio of A260/A280 = 1.80, and the concentration of ctDNA was determined by the absorption of ctDNA at 260 nm (ε260 = 6600 L mol−1 cm−1) [9]. Apigenin (>98%) was obtained from Shanghai Jingchun Reagent Limited Company (Shanghai, China). A stock

Absorption studies

Absorption spectra as a convenient technology are usually used to study the interaction of small molecules with nucleic acid. On account of the transition of n–π∗ and π–π∗ conjugated system, apigenin displays some spectrum characters. The peak wavelengths of apigenin were at 349 nm and 271 nm, respectively. Fig. 2 showed the absorption spectra of apigenin with the addition of ctDNA. With increasing the concentration of ctDNA, the absorbance at 349 nm decreased with a red shift from 349 nm to 351 nm,

Conclusion

In conclusion, the interactions of apigenin with ctDNA have been studied in Tris–HCl buffer solution (pH 7.4) using UV- and fluorescence spectroscopic techniques. The results indicated that the binding mode of apigenin to DNA is an intercalation binding, which was supported by the results from DNA melting studies and viscosity measurements. By the analysis of UV spectrum, it was observed that apigenin could slide into the base pairs when binding to ctDNA. The binding of apigenin to ctDNA is

Acknowledgements

The work was supported by National Natural Science Foundation of China (21205068) and Shandong Natural Science Foundation (ZR2010BL013).

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