Interaction of the flavonoid hesperidin with bovine serum albumin: A fluorescence quenching study

doi:10.1016/j.jlumin.2006.06.013

Journal of Luminescence

Volume 126, Issue 1, September 2007, Pages 211-218

https://doi.org/10.1016/j.jlumin.2006.06.013 Get rights and content

Abstract

The interaction between the flavonoid hesperidin and bovine serum albumin (BSA) was investigated by fluorescence and UV/Vis absorption spectroscopy. The results revealed that hesperidin caused the fluorescence quenching of BSA through a static quenching procedure. The hydrophobic and electrostatic interactions play a major role in stabilizing the complex. The binding site number n, and apparent binding constant K_A, corresponding thermodynamic parameters ΔG^o, ΔH^o, ΔS^o at different temperatures were calculated. The distance r between donor (BSA) and acceptor (hesperidin) was obtained according to fluorescence resonance energy transfer. The effect of Cu²⁺, Zn²⁺, Ni²⁺, Co²⁺, and Mn²⁺ on the binding constants between hesperidin and BSA were studied. The effect of hesperidin on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy and UV/Vis absorption spectroscopy.

Introduction

Polyphenols are secondary plant metabolites and have received much attention because of their potential health benefits [1], [2]. Flavonoids are natural substances with variable phenolic structures. More than 4000 flavonoids have been identified and grouped according to their molecular structures [3]. The best-described property of almost every group of flavonoids is their capacity to act as antioxidants able to scavenge free radicals and reactive oxygen species, which are associated with several forms of tissue damage and disease, including cancer and atherosclerosis, as well as with aging [3], [4], [5].

Hesperidin (Scheme 1), a flavanone-type flavonoid, is abundant in citrus fruit [3] and has been reported to exert a wide range of pharmacological effects [6]. Hesperidin can improve venous tone, enhance microcirculation, assist healing of venous ulcers and it is used for the treatment of chronic venous insufficiency, hemorrhoids and the prevention of postoperative thromboembolism [7].

Studies have showed that satsuma mandarin juice enriched with hesperidin has been shown to significantly reduce the incidence of colon cancer in rats [8], and orange juice was able to significantly inhibit atherosclerosis and lower cholesterol and triglycerides [9]. In view of the worldwide consumption of citric juice, it has been widely studied for the antioxidant activity of hesperidin in vitro [10], [11]. In addition, Yoshikawa et al. [12] reported that the double-strand breakage reaction in DNA molecules by cyanine dye is protected in the presence of glucosyl-hesperidin. Wilmsen et al. [3] reported that hesperidin was able to reduce significantly the level of the free radical DPPH (1,1–diphenyl-2-picrylhydrazyl) with similar efficacy of trolox and provide strongly cellular antioxidant protection against the damaging effects induced by paraquat and peroxide hydrogen.

Serum albumins are the major soluble protein constituents of the circulatory system and have many physiological functions [13]. The most important property of this group of proteins is that they serve as a depot protein and as a transport protein for many drugs and other bioactive small molecules [14]. Bovine serum albumin (BSA) has been one of the most extensively studied of this group of proteins, especially of its structural homology with human serum albumin (HSA) [13]. BSA is made up of three homologous domains (I, II, III), which are divided into nine loops (L1–L9) by 17 disulfide bonds. The loops in each domain are made up of a sequence of large–small–large loops forming a triplet. Each domain in turn is the product of two subdomains [1]. BSA has two tryptophans, Trp-134 and Trp-212, embedded in the first subdomain IB and subdomain IIA, respectively. HSA is a globular protein composed of 585 amino acid residues in three homologous α-helices domains (I–III). Each domain contains 10 helices and is divided into antiparallel six helices and four subdomains (A and B) [15]. There is only one tryptophan located at position 214 along the chain, in subdomain IIA of HSA. The molecular interactions of quercetin, rutin and other flavonoids with proteins have been investigated successfully [1], [16], [17], [18]. However, the molecular structure of hesperidin has difference compared with quercetin, rutin and other type of flavonoids, so it has different biological and pharmacological functions. Therefore, studying the interaction between the BSA and hesperidin is imperative and can obtain a lot of information of drug actions.

Fluorescence quenching is an important method to study the interaction of substances with protein because it is sensitive and relatively easy to use. Fluorescence spectroscopy is essentially a probe technique sensing changes in the local environment of the fluorophore, which distinguishes it from generalized techniques, such as calorimetry, far-ultraviolet circular dichroism (CD), and infrared (IR) spectroscopy. Also, various possibilities of structural rearrangements in the environment of the fluorophore may lead to a similar fluorescence signal; they can complicate interpretation of the experimental result and be exploited to obtain unique structural and dynamic information [19], [20], [21]. In the present work, we demonstrated the binding of hesperidin to BSA and the thermodynamics of their interaction. In order to attain these objectives, we planned to carry out detailed investigation of hesperidin–BSA association using fluorescence spectroscopy and UV/Vis absorption spectroscopy. The effect of Cu²⁺, Zn²⁺, Ni²⁺, Co²⁺, and Mn²⁺ on the binding constants between hesperidin and BSA were studied. Through fluorescence resonance energy transfer (FRET), synchronous fluorescence spectroscopy and UV/Vis absorption spectroscopy, we planned to further investigate the effect of the energy transfer and the effect of hesperidin on the conformation of BSA.

Section snippets

Materials

Hesperidin was purchased from Sigma (St. Louis, MO, USA), the purity of which is not less than 98%. BSA was purchased from Sino-Biotechnology Company (Shanghai, China), The buffer Tris was purchased from Acros (Geel, Belgium), and NaCl, HCl, etc. were all of analytical purity. BSA solution (1.0×10⁻⁶ mol L⁻¹) was prepared in pH 7.40 Tris-HCl buffer solution (0.05 mol L⁻¹ Tris, 0.1 mol L⁻¹ NaCl). The hesperidin solution (5.0×10⁻⁴ mol L⁻¹) was prepared in pH 7.40 Tris-HCl buffer containing 10% DMSO (v/v)

Fluorescence quenching

BSA molecule has two tryptophan residues that possess intrinsic fluorescence: Trp-134 in the first subdomain IB of the albumin molecule and Trp-212 in subdomain IIA. Trp-212 is located within a hydrophobic binding pocket of the protein and Trp-134 is located on the surface of the albumin molecule [27], [28]. A valuable feature of intrinsic fluorescence of proteins is the high sensitivity of tryptophan to its local environment [24]. Changes in emission spectra of tryptophan are common in

Conclusions

A fluorescence method for the rapid and simple determination of the interaction between hesperidin and BSA was provided. The method is easy to operate and is reliable, practical, and simple. Analysis was made in the present work using the date for protein fluorescence changes induced by flavonoid molecule. The results obtained give preliminary information on the binding of hesperidin to BSA. The results revealed the presence of a single class of binding site on BSA and its binding constants, K_A

Acknowledgments

We gratefully acknowledge financial support of Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection (JLCBE06032).

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Interaction of the flavonoid hesperidin with bovine serum albumin: A fluorescence quenching study

Abstract

Introduction

Section snippets

Materials

Fluorescence quenching

Conclusions

Acknowledgments

Am. J. Clin. Nutr.

J. Pharm. Biomed. Anal.

J. Pharm. Biomed. Anal.

Cancer Lett.

FEBS Lett.

Ann. Rep. Med. Chem.

J. Mol. Biol.

Biochim. Biophys. Acta

J. Mol. Struct.

J. Mol. Struct.

Spectrochim. Acta A

J. Mol. Struct.

Bioorg. Med. Chem.

J. Mol. Struct.

Bioorg. Med. Chem.

Bioelectrochemistry

J. Agric. Food. Chem.

Nutr. Res. Rev.

J. Agric. Food. Chem.

Arch. Pharm. Res.

Int. J. Cancer