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Latin American applied research

versión On-line ISSN 1851-8796

Lat. Am. appl. res. vol.42 no.2 Bahía Blanca abr. 2012

 

Study of the interaction of galangin, kaempferol and quercetin with BSA

Z. D. Fu†‡, X. Q. Chen and F. P. Jiao

College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, P.R. China.
Changsha Research Institute of Mining and Metallurgy, Changsha, Hunan 410012, P.R China
xqchen@mail.csu.edu.cn

Abstract — The interactions between Bovine Serum Albumin (BSA) and three flavonols, galangin, kaempferol and quercetin were studied by means of fluorescence spectroscopy. The fluorescence intensity of BSA exhibits remarkable decrease along with appreciable blue-shift of its maximum emission wavelength upon addition of the three compounds, respectively. The respective binding constant Ka and number of binding sites of each compound were calculated, and the quenching mechanism was proposed. Based on the values of thermodynamic parameters, the binding of each compound proceeds spontaneously with BSA. The binding distance between each and BSA was obtained by Foerster's dipole-dipole non-radiation energy transfer mechanism.

Keywords — Galangin; Kaempferol; Quercetin; Flavonols; Bovine Serum Albumin; Fluorescence Spectroscopy.

I. INTRODUCTION

The interaction between bio-macromolecules and drugs has attracted great interest among researchers since several decades (Xiang et al., 2008; Soares et al., 2007). Among bio-macromolecules, serum albumin is the most abundant protein in the circulatory system of man or animal, which carries plenty of drugs to all places of the body (Malonga et al., 2006). The drug-protein interaction may result in the formation of a stable protein-drug complex, which has important effect on the distribution, free concentration and the metabolism of drug in the blood stream (Xiao et al., 2008a). Therefore, studies on the binding of drug with protein will facilitate interpretation of the metabolism and transporting process of drug, and will help to explain the relationship between structures and functions of protein.

The interaction between molecules including hydrogen bonding, ionic and van der Waals interactions (Jiao et al., 2009). Protein-drug interactions play an important role in a variety of biological processes (Fuentes et al., 2007).

Flavonoids have been suggested to have several potential health benefits due to their antioxidant activities, which are attributed to the presence of phenolic hydroxyl moieties on the structure (Keli et al., 1996; Knekt et al., 1996).

Fluorescence spectroscopy is an appropriate method to determine the interaction between small molecules and biomacromolecules (Xiao et al., 2008b). By analyzing the fluorescence parameters, information concerning the structural changes in biomacromolecule can be obtained. There have been several studies on fluorescence quenching of proteins induced by flavonoids and other polyphenols (Riihimaki et al., 2008; Xiao et al., 2008c).

This paper studied the interaction between BSA and three flavonols, galangin, kaempferol and quenching by fluorescence spectroscopy, and compared the difference of those reactions.

II. MATERIALS AND METHODS

A. Apparatus

The fluorescence spectra were recorded on a JASCO FP-6500 spectrofluorometer equipped with a thermostated compartment using 1.0cm quartz cuvette. The pH measurements were carried out on a PHS-3C Exact Digital pH meter, which was calibrated with standard pH buffers.

B. Reagents

BSA (V, Sigma) diluted into 1.0×10-5 mol⋅L-1 as the reserving solution; galangin, kaempferol, quercetin (Shan-hai u-sea biotech co., ltd. purity>99%) were dissolved into the mixture of methanol and water, whose volume ratio is 1: 1, then diluted into 1.0×10-4 mol L-1 as the reserving solution. Tris-HCl buffer (0.20 mol/L, pH 7.4) containing 0.10 mol/L NaCl was selected to keep the pH value and maintain the ionic strength of the solution. All the reagents used in this experiment were analytical grade, and the water was newly doubly-distilled and deionized.

C. Fluorescence spectrum analysis

1.0 mL BSA solution and the volume of flavonol solution indicated in Fig. 1 were added into 10 mL volumetric flasks respectively. After diluting to 10 mL with deionized water and mixing thoroughly, each flask was kept in a constant temperature water bath at 290 K, 300 K and 310 K for 1h. Fixed λex at 280 nm, each solution was scanned to determine the fluorescence intensity in the range of 290nm to 450 nm. Then absorption spectrum of each compound solution (1.0×10-6 mol⋅L-1) was scanned in the range of 250 nm to 400 nm.


Fig. 1: Effects of galangin (A), kaempferol (B) and Quercetin (C) on fluorescence spectra of BSA at 37 °C.c (BSA) = 1.0× 10-6 mol⋅L-1, c (Flavonols ) = 1× 10-5 mol⋅L-1, a-k: 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0µl.

III. RESULTS AND DISCUSSION

A. Characteristics of fluorescence spectra

Fluorescence quenching spectra of BSA in the presence of various concentrations of galangin, kaempferol and quercetin are shown in Fig.1. With the increase of the concentration of galangin, kaempferol and quercetin, the fluorescence of BSA were quenched regularly, and the maximum emission wavelengths of three flavonols have a slight blue shift. The results show that galangin, kaempferol and quercetin have a reaction with BSA, making a change of the microenvironment of at least one of the two indole rings in the BSA (Tian et al., 2004). Complexes would be generated from three flavonols and BSA, which have little or no fluorescence.

B. Type of the fluorescence quenching

The system of quenching can be divided into static quenching and dynamic quenching. The dynamic quenching is an interaction process between quencher and excited state molecule of the fluorescent substance, which follows the Stern-Volmer equation (Yan et al., 2005). The Eq. (1) represents the fluorescence intensity of the fluorescent substance.

(1)

where F0 and F are the fluorescence intensities before and after the addition of the quencher, Kq the rate constant of bimolecular quenching, τ0 the average lifetime of the electronically excited state of BSA in the absence of quencher, KSV the kinetic quenching constant, [Q] the concentration of the quencher.

Dynamic quenching was assumed as the type of the fluorescence quenching between three flavonols and BSA, so the process will accord with the equation above. Figure 2 shows the Stern-Volmer curves in different temperature plotted with F0/F as ordinate against [Q] as abscissa. The figure shows the curves were linear, and with the temperature increased, all of the slopes decreased. According to the Stern-Volmer equation, Kq and KSV were calculated and shown in Table 1.


Fig. 2: Stern-Volmer curves of BSA quenched by galangin (a), kaempferol (b) and Quercetin (c).

Table 1: The quenching constants between BSA and flavones

The results show that the KSV decreases with increasing temperature, indicating that the probable quenching mechanism of fluorescence of BSA by three flavonols is a static quenching procedure, resulting in forming flavonols-BSA complexs and the stability of the complex decreases with increasing temperature.

According to the literatures (Lien et al., 1999; Chenetal, 1996), for dynamic quenching, the lifetime of biological macromolecules was generally about 1×10-8 s, the quenching constant of maximum diffusion collision was generally 2.0×1010 L⋅mol-1⋅s-1, and the KSV was increased with the temperature of the system increased. Obviously, the quenching rate constants (KQ) of three flavonols were far more than 1×10-8 s, and KSV was decreased with the temperature increased. This means that the quenching is static quenching procedure by forming conjugates of adduct.

C. Calculation of binding constant

In the process of static quenching, the relation of quenching constant, fluorescence intensity and concentration of the quencher can be described as follow (Nemethy and Scheraga, 1962):

(2)

where is the binding constant and n the number of binding sites of the biomacromolecule. According to the plot with the left side of the equation as ordinate against logarithm of [Q] as abscissa, the value of and n were obtained and shown in Table 2. The high value of indicates there is a strong interaction force between three flavonols and BSA. With the number of hydroxy on the B-ring of flavonols increased, and n increased. These results show the hydroxy on the B-ring of flavonols will strengthen the binding capacity of the small molecules. So it can be guessed that the hydroxy on the B-ring of flavonols participates in the reaction and becomes the important groups. Some literature reports the 3'-OH and 4'-OH on the B-ring of flavonols will enhance the antioxidant and eliminate free radical ability, which can be attributed to their high binding capacity (Liu et al., 2010; Xiao et al., 2008a).

Table 2: The binding constants of BSA with flavones

D. Binding mode of the Galangin, Kaempferol and Quercetin with BSA

The binding force between the drug molecules and protein may involve hydrophobic forces, Van Der Waals interactions, electrostatic interactions and hydrogen bonds, etc. According to the data of enthalpy change (ΔH) and entropy change (ΔS), the model of interaction between drug and biomolecule can be concluded (Ross and Subramanian, 1981): (1) ΔH>0, ΔS>0, hydrophobic forces; (2) ΔH<0, ΔS>0, electrostatic interactions. When the change of temperature is not much, the ΔH can be regard as a constant. The thermodynamic parameters were calculated by the first law of thermodynamics and the binding constant of each compound at different temperature is shown in Table 3. From the results shown in Table 3, it can be found DG of every compound is less than zero, this shows the reaction is carried out by itself and hydrophobic forces is the major force.

Table 3: The thermodynamic parameters of the interactions between BSA and flavones

E. Binding distance

According to Foerster's dipole-dipole non-radiation energy transfer mechanism, in the similar concentration of donor and acceptor condition, if the fluorescence spectrum of donor and UV of receptor have enough overlap, and the distance between them is less than 7nm, it is likely to occur Non-energy radiation between donor and recipient, resulting in donor fluorescence quenching.

The binding distance between galangin, kaempferol, quercetin and BSA can be calculated with the equation:

(3)

where F0 and F are the fluorescence intensities with and without the existent of receptor, r the distance between the donor and receptor, R0 the critical distance when energy transfer efficiency was 50%. The value of R0 can be calculated with equation

(4)

where K2 is dipole orientation factor, N the refractive index of the medium in the system, Φ the fluorescence quantum yield of donor, J the integral area of the overlapped spectrums between the fluorescence spectrums of donor and the ultraviolet absorption spectrum. J can be calculated with the equation:

(5)

where F(λ) and ε(λ) are the fluorescence intensity of the donor and the molar absorptivity when the wavelength is λ. In most conditions, K2, N and Φ is respectively 2/3, 1.336 and 0.118.

Figure 3 shows the fluorescence spectra of BSA and the ultraviolet absorption spectra of galangin, kaempferol and quercetin. With the equations mentioned, J, R0, E, r were calculated and shown in Table 4. The binding distance of the galangin, kaempferol, quercetin and BSA is respectively 3.38 nm, 3.64 nm and 3.75 nm. All the distances are less than 7 nm, this indicates there is an energy transfer between three flavonols and BSA, and the static fluorescence quenching of BSA is caused by non-radiation energy transfer.


Fig. 3: The overlaps of the absorption spectra of galangin (a), kaempferol (b), quercetin (c) UV and fluorescence spectra of BSA. BSA, 1.0 × 10-6 mol⋅ L-1; Flavonols, 1.0 × 10-6 mol⋅ L-1

Table 4: The distance parameter of BSA with flavones

F. Conformation investigation

To explore the structural change of BSA by addition of three flavonols, we measured the synchronous fluorescence spectra (Fig. 4) of BSA with various concentration of three flavonols. Synchronous fluorescence is a kind of simple and effective means to measure the fluorescence quenching and the possible shift of the maximum emission wavelength λmax, relative to the alteration of the polarity around the chromophore microenvironment. Δλ, representing the value of difference between excitation and emission wavelengths, is an important operating parameter. When the Δλ is 15 nm, the synchronous fluorescence spectrometry shows the spectral characters of tyrosine residues, and when the Δλ is 60 nm, tryptophan residues are shown. When Δλ is set at 15 or 60 nm the shift of the λmax and the fluorescence quenching of BSA imply the alteration of polarity microenvironment around Tyr or Trp residues and the state of drug binding to BSA.

Fig.4: The synchronous fluorescence spectrometry of galangin (1, 2), kaempferol (3, 4), quercetin (5, 6) and BSA. Δλ=15nm (A), Δλ=60nm (B), [BSA]=1.0×10-6 mol⋅L-1; [galangin]=1×10-5 mol⋅L-1; a-f: 0,100,200,300,400,500 µL of galangin. [kaempferol]=1×10-5 mol⋅L-1; a-f: 0,100,200,300,400,500 µL of kaempferol.

The obviously fluorescence quenchings in two situations show three flavonols, tyrosine and tryptophan residues can bind simultaneously (Fig. 4). However, when the Δλ is 60 nm, the fluorescence decline is much larger than 15 nm. It can be concluded that three flavonols are closer with tryptophan or have higher efficiency of energy transfer compared with tyrosine. To tyrosine residues, there is a slight blue shift of the emission wavelength, which shows the interaction changes the microenvironment of tyrosine to an embedding state. While to tryptophan residues, there is a slight red shift of the maximum emission wavelength, which shows the interaction changes the microenvironment of tryptophan to an exposed state.

IV. CONCLUSION

In this paper, the interaction between three flavonols (galangin, kaempferol and quercetin) and BSA was studied by fluorescence spectroscopy. The experimental results indicated that the probable quenching mechanism of fluorescence of BSA by three flavonols is a static quenching procedure and the binding reaction is spontaneous. The binding force is largely mediated by hydrophobic forces. The results obtained from synchronous fluorescence spectra show that the structure of BSA molecules is changed dramatically in the presence of three flavonols. The fluorescence intensity of BSA exhibits remarkable decrease along with appreciable blue-shift of its maximum emission wavelength upon addition of the three compounds, respectively.

ACKNOWLEDGEMENTS
We wish to acknowledge the support given to this work by the China National Natural Science Foundation (project No. 20805058) and China Postdoctoral Science Foundation (Project No. 20080431023).

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Received: November 11, 2009.
Accepted: August 22, 2011.
Recommended by Subject Editor Ana Lea Cukierman.