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Interactions between flavonoids and hemoglobin in lecithin liposomes
International Journal of Biological Macromolecules 40 (2007) 305–311
Interactions between flavonoids and hemoglobin in lecithin liposomes Juqun Xi, Rong Guo ∗ School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, PR China Received 12 February 2006; received in revised form 19 August 2006; accepted 21 August 2006 Available online 26 August 2006
Abstract In this paper, the binding of flavonoids (quercetin and rutin) to hemoglobin (Hb) have been investigated by fluorescence, absorption spectroscopy and circular dichroism (CD) spectroscopy. The binding parameters and binding mode between flavonoids and Hb are determined and the results of CD and synchronous fluorescence spectra indicate a conformational change of Hb with addition of flavonoids. The effects of lecithin liposomes on the binding parameter of quercetin and rutin to Hb are also studied. When incorporated into liposome, flavonoids can reduce the fluorescence of tryptophanyl residues of Hb to a lesser extent. The difference of the structure characteristics between quercetin and rutin has a significant effect on their binding affinity for Hb. © 2006 Elsevier B.V. All rights reserved. Keywords: Rutin; Quercetin; Hemoglobin; Liposome; Fluorescence quenching; Circular dichroism
1. Introduction Polyphenols are secondary plant metabolites and have received much attention because of their potential health benefits [1–3]. Flavonoids, regular constituents of the diet, were first identified as Vitamin P, and along with Vitamin C were found to be important in the maintenance of capillary wall integrity and capillary resistance. They respond to light and are known to control the level of auxins, the regulators of plant growth and differentiation. Consumption of plants and plant products that are rich in flavonoids, such as cocoa, wine, tea, and berries, has been related with protective effects against cardiovascular disease and certain forms of cancer [4,5]. They have been found to act as free-radical scavengers and have been widely studied for their antioxidant activity in vitro. However, the reality about their in vivo activity still remains uncertain, and questions concerning their absorption, metabolism, and bioavailability are still unanswered [6–8]. Current knowledge suggests that factors such as protein binding may impair polyphenol absorption and bioavailability and even mask their antioxidant activity [9,10]. Protein–polyphenol association is a well-known phenomenon; however, it is only relatively recently that any considerable information has been obtained in the area of how the structure of either the protein or the polyphenol may affect the interaction. ∗
Corresponding author. Tel.: +86 514 7975219; fax: +86 514 7311374. E-mail address: [email protected] (R. Guo).
0141-8130/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2006.08.011
In this paper, we study the association of flavonoids with hemoglobin (Hb) and the effects of liposomes on the binding of flavonoids to Hb. Liposomes are vesicles in which a small volume is entirely enclosed by a membrane composed of phospholipids [11–13]. Proteins interact with the lipid bilayer by binding or adsorption to its surface, insertion into the hydrocarbon region of membrane interior or penetration through the membrane. The experiment is designed to test the possibility of entrapping the flavonoids (quercetin and rutin, Fig. 1) into liposome for a better study of the interactions between drugs and proteins in simulating life system. In addition, the regularity of the binding properties of the drug to Hb is investigated from the structure characteristics angles. The work is worthy doing and benefits the understanding of the transports and medicine process for the flavonoids in Chinese herbal medicine. The knowledge of the structure features that determine the binding capacity of the compounds and protein may open up new avenues for the design of the most suitable flavonoids derivatives with structural variants. 2. Materials and method 2.1. Reagents The regents used were Lecithin (Microorganism Culture Medium Products Refinery of Haidian District, China), Hemoglobin (Shanghai Lizhu Dongfeng Bioligy Technique
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J. Xi, R. Guo / International Journal of Biological Macromolecules 40 (2007) 305–311
3. Results and discussions 3.1. Flavonoids induced quenching mechanism of Hb fluorescence Hb has a molecular weight of approximately 67,000 and contains two α and two β subunits, each of which has one redoxiron heme as its prosthetic group, the heme is located in crevices at the exterior of the subunit [14]. Due to tryptophan (Trp), Hb shows an emission peak at 330 nm with the excitation wavelength at 280 nm. An intrinsic fluorescence study is performed to evaluate changes in tertiary structure caused by reaction of Hb with flavonoids (quercetin and rutin). The effect of drug on Hb fluorescence intensity is shown in Fig. 2. The fluorescence intensity of Hb is gradually decreased when the solution of flavonoids is added, which indicates that flavonoids can bind to Hb. The fluorescence quenching data are analyzed by the Stern–Volmer equation [15]: Fig. 1. Structures of (a) quercetin and (b) rutin.
Ltd., China), Quercetin (China Drug Biological Products Qualifying Institute, China), Rutin (China Drug Biological Products Qualifying Institute, China). Water used was distilled twice. 2.2. Preparation of liposomes A chloroform solution of lecithin was placed in a 50 mL round-bottomed flask. Then the solvent was removed under vacuum at 30 ◦ C by using a rotary evaporator. The resulting thin film of lecithin on the walls of the flask was dried under vacuum at room temperature for at least 2 h. Then the dry lecithin film was dispersed in phosphate buffered saline (PBS, pH 7.4) and was sonicated for 2 h using a CQ250 water bath-type sonicator (220 V, 50 Hz, Shanghai Ultrasonic Wave Instrument Works, China) to obtain liposomes. 2.3. Fluorescence quenching measurement The fluorescence intensities of Hb were recorded with an RF4550 PC spectrophotometer (Hitachi Corporation, Japan). The excitation and emission wavelength for Hb were 280 nm and 330 nm. 2.4. UV–vis and CD spectra measurement The flavonoids solutions with various concentrations were added to the Hb solutions. After about 1 h at 25 ± 0.1 ◦ C for reaching the equilibrium, JASCO model 810 spectropolarimeter (Japan) was used to record the CD spectra from 190 nm to 300 nm. The absorption spectra of flavonoids in liposomal system were measured by using the UV-2501 spectrophotometer (Shimadzu Corporation, Japan) in the range of the wavelength 500–200 nm.
F0 = 1 + KSV [Q] F
(1)
where F0 and F are the fluorescence intensities before and after the addition of the quencher, respectively. [Q] is the concentration of the quencher, and KSV is the Stern–Volmer quenching constant. As Table 1 showed, the quenching constant decreases with increasing temperature, a characteristic that coincides with the static type of quenching mechanism. The quenching constant can be interpreted as the association constant or binding constant of the complexation reaction because static quenching arises from the formation of a dark complex between the fluorophore and the quencher [16]. 3.2. Binding parameters Flavonoids induced fluorescence quenching data of Hb are analyzed to obtain various binding parameters. The binding constant (K) and binding affinity (n) can be calculated using the equation for the static quenching process [17]: F0 − F log = log K + n log[Q] (2) F A plot of log[(F0 − F)/F] versus log[Q] gives a straight line (Fig. 3) using least-squares analysis whose slope is equal to n (binding affinity) and the intercept on Y-axis to log K. The K and n at 25 ◦ C and 35 ◦ C are listed in Table 1 for comparison. The binding constant of rutin is lower than that of quercetin. These results are may related to the structures of the two flavonoids (Fig. 1). Rutin is a glucoside of quercetin, incorporating the disaccharide rutinose. The presence of rutinose renders rutin less hydrophobic than quercetin and also more bulky. This latter fact may result in steric hindrance of its penetration of the hydrophobic pocket of Hb and may generally affect the orientation of the rutin molecules in relation to the protein. So the binding capacity of rutin with protein is less than that of quercetin. These results are agreed with the former studies of Bi et al. [18].
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Table 1 Binding and thermodynamic parameters of flavonoids–Hb interactions (a) quercetin and (b) rutin T (K)
Ksv (×104 L mol−1 )
K (×105 L mol−1 )
n
G (kJ mol−1 )
H (kJ mol−1 )
S (J mol−1 )
(a) Quercetin 298 308
5.34 4.71
6.02 2.51
1.28 1.19
−32.97 −31.84
−66.76
−113.39
(b) Rutin 298 308
5.12 2.34
2.34 0.87
1.16 1.05
−30.63 −29.14
−74.98
−149.83
3.3. Synchronous fluorescence and CD spectra To explore the structural change of Hb by addition of flavonoids, we measured synchronous fluorescence spectra (Fig. 4) of Hb with various amounts of flavonoids. The synchronous fluorescence spectra give information about the molecular environment in the vicinity of the chromosphere molecules. Yuan et al. [19] suggested a useful method to study the environment of amino acid residues by measuring the possible shift in wavelength emission maximum λmax , and the shift in position of emission maximum corresponding to the changes of the polarity around the chromophore molecule. When the D-value (λ) between excitation wavelength and emission wavelength are stabilized at 30 nm or 60 nm, the synchronous fluorescence gives the characteristic information of tyrosine residues or tryptophan residues [20]. The effects of flavonoids on Hb synchronous fluorescence spectroscopy are shown in Fig. 4 (date for quercetin not shown). It is apparent from Fig. 4 that the little stronger red shift of tryptophan residues exhibits fluorescence upon addition of rutin, whereas the emission maximum of tyrosine kept the position. The red shift of the emission maximum indicates that the conformation of Hb is changed and the polarity around the tryptophan residues is increased and the hydrophobicity is decreased.
Fig. 2. Fluorescence spectra of Hb in the prescence of (a) quercetin and (b) rutin at 25 ◦ C. Total concentrations of quercetin and rutin: (1) 0.0 mol L−1 ; (2) 0.5 × 10−5 mol L−1 ; (3)1.0 × 10−5 mol L−1 ; (4) 1.5 × 10−5 mol L−1 ; (5) 2.0 × 10−5 mol L−1 ; (6) 2.5 × 10−5 mol L−1 ; (7) 3.0 × 10−5 mol L−1 ; (8) 3.5 × 10−5 mol L−1 . The concentration of Hb is 3.0 × 10−6 mol L−1 .
Fig. 3. The plots of log[Q] vs. log((F0 − F)/F) at 25 ◦ C for (1) rutin and (2) quercetin. The concentration of Hb is 3.0 × 10−6 mol L−1 .
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Fig. 5. Synchronous fluorescence spectra of Hb in the prescence of rutin at 25 ◦ C: (a) λ = 30 nm; (b) λ = 60 nm. Total concentrations of rutin: (1) 0.0 mol L−1 ; (2) 1.0 × 10−5 mol L−1 ; (3) 1.5 × 10−5 mol L−1 ; (4) 2.0 × 10−5 mol L−1 ; (5) 2.5 × 10−5 mol L−1 ; (6) 3.0 × 10−5 mol L−1 ; (7) 3.5 × 10−5 mol L−1 . The concentration of Hb is 3.0 × 10−6 mol L−1 .
in the presence and absence of flavonoids are similar in shape, indicating that the structure of Hb after the addition of drug is also predominantly -helical. From these results, it is apparent that interaction of flavonoids with Hb causes a conformational change of the protein with the loss of helical stability. 3.4. Mode of binding There are essentially four types of noncovalent interactions that could play a role in ligand binding to proteins. These are hydrogen bonds, van der Waals forces, hydrophobic bonds, and electrostatic interactions [22]. If the enthalpy changes (H◦ ) do not vary significantly over the temperature range studied, then its value and that of S◦ can be determined from the Van’t Hoff equation: Fig. 4. Changes of the CD spectrum of Hb upon the addition of rutin at 298 K. Rutin to Hb (3.0 × 10−6 mol L−1 ) ratios: (1) 0:1, (2) 4:1, and (3) 8:1.
To gain a better understanding in physicochemical properties of flavonoid governing its spectral behavior and to draw relevant conclusions on the flavonoid–Hb binding mechanism, additional CD measurements were performed on flavonoid and the flavonoid–Hb complex. Take the binding of rutin molecule to Hb for example (data for quercetin not shown), Fig. 5 shows the CD spectra of the Hb and Hb–rutin complex obtained at pH 7.4. The CD spectra of Hb exhibited two negative minima at 209 nm and 221 nm, which is typical characterization of the helix structure of class proteins [21]. The binding of rutin to Hb decreases both of these bands, clearly indicating the decrease of the -helical content in protein. However, the CD spectra of Hb
ln K = −
S ◦ H ◦ + RT R
(3)
where K is the binding constant at the corresponding temperature and R is the gas constant. The equation provides the enthalpy (H◦ ) and entropy (S◦ ) changes on binding. The free energy of binding is estimated from the following relationship: G◦ = H ◦ − TS ◦
(4)
To obtain such information, the implications of the present results have been discussed in conjunction with thermodynamic characteristics obtained for flavonoids binding, and the thermodynamic parameters are listed in Table 1. The negative sign for G◦ indicates the spontaneity of the binding of flavonoids with Hb. Based on the characteristic signs of the thermodynamic
J. Xi, R. Guo / International Journal of Biological Macromolecules 40 (2007) 305–311
309
parameters at the various interactions [23], the negative H◦ and S◦ values for flavonoids binding to Hb are associated with hydrogen bonding and van der Waals interaction. 3.5. The binding parameters on liposomal system 3.5.1. The distribution of flavonoids between the lecithin liposome and the aqueous phase UV spectra of flavonoids exhibit two major absorption bands in the region 200–400 nm. Band-I (300–380 nm) is considered to be associated with absorption due to the ring-B cinnamoyl system and band-II (200–280 nm) is considered to be associated with absorption due to the ring-A benzoyl system [24]. Fig. 6(a and b) shows the spectra of quercetin and rutin in water and lecithin liposomes at 25 ◦ C, respectively. In lecithin liposome, the absorbance at 340 nm increases and the band exhibits red shifts. The UV spectra of the flavonoids shift towards higher wavelength indicating that great numbers of flavonoid molecules are being incorporated into the liposomes. The flavonoid molecule has aromatic rings, its solubility in water is low and it exists in the anionic state with one or two charges. Therefore, quercetin and rutin will be easily extracted into the lecithin liposomes through hydrophobic interaction. Moreover, the lecithin molecule does carry a positive charge group due to the presence of N+ (CH3 )3 , it will definitely try to aggregate with flavonoid’s anion and this promotes the process of the location of flavonoids in liposome. The distribution coefficient KD of querctein and rutin between the lecithin liposome and the aqueous phase can be formulated as KD =
flavonoidL flavonoidW
(5)
Here flavonoidL and flavonoidW are the mole fractions of flavonoid in the lecithin liposome and aqueous phases, respectively. The distribution coefficient KD can be calculated by the following equation [25,26]: 1 1 1 1 × + = εΨ − ε W KD (εL − εW ) C εL − εW
(6)
Here C is the concentration of lecithin, ε the apparent molar absorptivity of flavonoids at given wavelength, εL and εm are the apparent molar absorptivities of flavonoids in the lecithin liposomes and aqueous phases, respectively. From the plot of 1/(ε − εW ) against 1/C, the distribution coefficient KD can be calculated from the slope and the intercept of the straight line (Fig. 7). The values of KD for quercetin and rutin are 1315.3 and 194.6, respectively. The distribution coefficient of quercetin is larger than that of rutin. This suggests the lecithin liposomes could incorporate more quercetin molecules. This result is probably concerned with the molecule structures of the two flavonoids. The presence of rutinose renders rutin molecule less hydrophobic than quercetin and also more bulky. These facts may result in steric hindrance of rutin incorporation into the hydrophobic region of liposome and may generally affect the concentrations of flavonoids in liposome.
Fig. 6. The absorption spectra of (a) quercetin and (b) rutin (3.0 × 10−5 mol L−1 ) in lecithin liposomes at 25 ◦ C. Lecithin concentration: (1) 0 g mL−1 , (2) 1.5 × 10−4 g mL−1 , and (3) 2.5 × 10−4 g mL−1 .
3.5.2. The effect of liposomes on the binding constants between the flavonoids and Hb Our former work shows that Hb can be adsorbed on lecithin liposomes by the hydrophobic interaction between Hb and lecithin molecules, which results in an increase of the microenvironmental polarity (I1 /I3 ) of pryene, a decrease of membrane fluidity (P), and microhydrophobicity of lecithin liposomes. With the increasing content of Hb, the permeability of the liposomal bilayer membranes increases [27]. These studies provide the foundation for the research of binding studied between flavonoids and protein in liposomes.
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Table 2 The effect of liposomes on the binding parameters between flavonoids and Hb at 289 K Compound
CHb (×10−6 mol L−1 )
CLecithin (×10−4 mol L−1 )
K (L mol−1 )
K /K
L Quercetin
3.0 3.0 1.0 1.0
1.0 2.5 1.0 2.5
7.19 × 104 5.01 × 104 1.65 × 105 1.14 × 105
0.12 0.08 0.27 0.19
Rutin
3.0 3.0 1.0 1.0
1.0 2.5 1.0 2.5
8.34 × 104 6.02 × 104 1.03 × 105 8.58 × 104
0.33 0.26 0.44 0.37
In the liposomal system, the fluorescence intensity of Hb also drops with the increase of flavonoids concentrations and the binding constants are determined at 25 ◦ C in the presence of lecithin liposome. The results shown in Table 2 reveal that the binding constants of the flavonoids with Hb are 8–44% of those without liposomes. This implies that when the two flavonoids are incorporated into liposome, they quench the fluorescence of the tryptophanyl residues of Hb to a lesser extent. In addition, the binding constant between quercetin and Hb (3.0 × 10−6 mol L−1 ) is reduced to 8% in liposomal system (2.5 × 10−4 g mol−1 ), but only to 26% for rutin in the same conditions. These results are due to the fact that the distribution coefficient of quercetin between the lecithin liposome and the aqueous phase is larger than that of rutin. A larger incorporated amount of quercetin into liposome induces the quench reaction between drug and protein to a lesser extent, so this difference in distribution coefficient of quercetin and rutin appears to have a significant effect on their binding affinity for Hb. Table 2 shows that the binding capacities of quercetin and rutin to Hb are greater at a higher liposome concentration (2.5 × 10−4 g mL−1 ) than those at lower liposome concentration
(1.0 × 10−4 g mL−1 ). This is due to the fact that the increase of the liposome concentration can bring more quercetin and rutin molecules entrapped into liposomal bilayer membranes, which can cause a lesser extent of the quenching reaction. From Table 2, it can also be seen that at higher Hb concentration (3.0 × 10−6 mol L−1 ), the binding constants of flavonoids with Hb are enhanced. These results are considered to be that the permeability of the liposomal bilayer membranes increases with the increasing content of Hb [27]. The increasing permeability of liposome at higher concentration of Hb enhances the amounts of quercetin and rutin molecules in aqueous phase, thus binding reactions between flavonoids and Hb become easier and the binding constants of quercetin and rutin to Hb are both increased. 4. Conclusions In this paper, the binding properties of two flavonoids (quercetin and rutin) to Hb were characterized by measuring the fluorescence and CD spectra. The results indicated a conformational change of Hb with addition of flavonoids. The binding parameters for flavonoids were obtained and the negative H◦ and S◦ values of thermodynamic parameters for flavonoids binding to Hb were associated with hydrogen bonding and van der Waals interaction. When incorporated into liposomes, quercetin and rutin could quench the fluorescence of the tryptophanyl residues of Hb to a lesser extent. However, the difference of the structure characteristics between them appears to have a significant effect on their incorporation amount in liopsome and the binding affinity for Hb. Acknowledgement This work is supported by National Natural Scientific Foundation of China (No. 20233010). References
Fig. 7. Plotting of (ε − εw)−1 vs. C−1 . The concentrations of (a) quercetin and (b) rutin are 3.0 × 10−5 mol L−1 .
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Interactions between flavonoids and hemoglobin in lecithin liposomes Juqun Xi, Rong Guo ∗ School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, PR China Received 12 February 2006; received in revised form 19 August 2006; accepted 21 August 2006 Available online 26 August 2006
Abstract In this paper, the binding of flavonoids (quercetin and rutin) to hemoglobin (Hb) have been investigated by fluorescence, absorption spectroscopy and circular dichroism (CD) spectroscopy. The binding parameters and binding mode between flavonoids and Hb are determined and the results of CD and synchronous fluorescence spectra indicate a conformational change of Hb with addition of flavonoids. The effects of lecithin liposomes on the binding parameter of quercetin and rutin to Hb are also studied. When incorporated into liposome, flavonoids can reduce the fluorescence of tryptophanyl residues of Hb to a lesser extent. The difference of the structure characteristics between quercetin and rutin has a significant effect on their binding affinity for Hb. © 2006 Elsevier B.V. All rights reserved. Keywords: Rutin; Quercetin; Hemoglobin; Liposome; Fluorescence quenching; Circular dichroism
1. Introduction Polyphenols are secondary plant metabolites and have received much attention because of their potential health benefits [1–3]. Flavonoids, regular constituents of the diet, were first identified as Vitamin P, and along with Vitamin C were found to be important in the maintenance of capillary wall integrity and capillary resistance. They respond to light and are known to control the level of auxins, the regulators of plant growth and differentiation. Consumption of plants and plant products that are rich in flavonoids, such as cocoa, wine, tea, and berries, has been related with protective effects against cardiovascular disease and certain forms of cancer [4,5]. They have been found to act as free-radical scavengers and have been widely studied for their antioxidant activity in vitro. However, the reality about their in vivo activity still remains uncertain, and questions concerning their absorption, metabolism, and bioavailability are still unanswered [6–8]. Current knowledge suggests that factors such as protein binding may impair polyphenol absorption and bioavailability and even mask their antioxidant activity [9,10]. Protein–polyphenol association is a well-known phenomenon; however, it is only relatively recently that any considerable information has been obtained in the area of how the structure of either the protein or the polyphenol may affect the interaction. ∗
Corresponding author. Tel.: +86 514 7975219; fax: +86 514 7311374. E-mail address: [email protected] (R. Guo).
0141-8130/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2006.08.011
In this paper, we study the association of flavonoids with hemoglobin (Hb) and the effects of liposomes on the binding of flavonoids to Hb. Liposomes are vesicles in which a small volume is entirely enclosed by a membrane composed of phospholipids [11–13]. Proteins interact with the lipid bilayer by binding or adsorption to its surface, insertion into the hydrocarbon region of membrane interior or penetration through the membrane. The experiment is designed to test the possibility of entrapping the flavonoids (quercetin and rutin, Fig. 1) into liposome for a better study of the interactions between drugs and proteins in simulating life system. In addition, the regularity of the binding properties of the drug to Hb is investigated from the structure characteristics angles. The work is worthy doing and benefits the understanding of the transports and medicine process for the flavonoids in Chinese herbal medicine. The knowledge of the structure features that determine the binding capacity of the compounds and protein may open up new avenues for the design of the most suitable flavonoids derivatives with structural variants. 2. Materials and method 2.1. Reagents The regents used were Lecithin (Microorganism Culture Medium Products Refinery of Haidian District, China), Hemoglobin (Shanghai Lizhu Dongfeng Bioligy Technique
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3. Results and discussions 3.1. Flavonoids induced quenching mechanism of Hb fluorescence Hb has a molecular weight of approximately 67,000 and contains two α and two β subunits, each of which has one redoxiron heme as its prosthetic group, the heme is located in crevices at the exterior of the subunit [14]. Due to tryptophan (Trp), Hb shows an emission peak at 330 nm with the excitation wavelength at 280 nm. An intrinsic fluorescence study is performed to evaluate changes in tertiary structure caused by reaction of Hb with flavonoids (quercetin and rutin). The effect of drug on Hb fluorescence intensity is shown in Fig. 2. The fluorescence intensity of Hb is gradually decreased when the solution of flavonoids is added, which indicates that flavonoids can bind to Hb. The fluorescence quenching data are analyzed by the Stern–Volmer equation [15]: Fig. 1. Structures of (a) quercetin and (b) rutin.
Ltd., China), Quercetin (China Drug Biological Products Qualifying Institute, China), Rutin (China Drug Biological Products Qualifying Institute, China). Water used was distilled twice. 2.2. Preparation of liposomes A chloroform solution of lecithin was placed in a 50 mL round-bottomed flask. Then the solvent was removed under vacuum at 30 ◦ C by using a rotary evaporator. The resulting thin film of lecithin on the walls of the flask was dried under vacuum at room temperature for at least 2 h. Then the dry lecithin film was dispersed in phosphate buffered saline (PBS, pH 7.4) and was sonicated for 2 h using a CQ250 water bath-type sonicator (220 V, 50 Hz, Shanghai Ultrasonic Wave Instrument Works, China) to obtain liposomes. 2.3. Fluorescence quenching measurement The fluorescence intensities of Hb were recorded with an RF4550 PC spectrophotometer (Hitachi Corporation, Japan). The excitation and emission wavelength for Hb were 280 nm and 330 nm. 2.4. UV–vis and CD spectra measurement The flavonoids solutions with various concentrations were added to the Hb solutions. After about 1 h at 25 ± 0.1 ◦ C for reaching the equilibrium, JASCO model 810 spectropolarimeter (Japan) was used to record the CD spectra from 190 nm to 300 nm. The absorption spectra of flavonoids in liposomal system were measured by using the UV-2501 spectrophotometer (Shimadzu Corporation, Japan) in the range of the wavelength 500–200 nm.
F0 = 1 + KSV [Q] F
(1)
where F0 and F are the fluorescence intensities before and after the addition of the quencher, respectively. [Q] is the concentration of the quencher, and KSV is the Stern–Volmer quenching constant. As Table 1 showed, the quenching constant decreases with increasing temperature, a characteristic that coincides with the static type of quenching mechanism. The quenching constant can be interpreted as the association constant or binding constant of the complexation reaction because static quenching arises from the formation of a dark complex between the fluorophore and the quencher [16]. 3.2. Binding parameters Flavonoids induced fluorescence quenching data of Hb are analyzed to obtain various binding parameters. The binding constant (K) and binding affinity (n) can be calculated using the equation for the static quenching process [17]: F0 − F log = log K + n log[Q] (2) F A plot of log[(F0 − F)/F] versus log[Q] gives a straight line (Fig. 3) using least-squares analysis whose slope is equal to n (binding affinity) and the intercept on Y-axis to log K. The K and n at 25 ◦ C and 35 ◦ C are listed in Table 1 for comparison. The binding constant of rutin is lower than that of quercetin. These results are may related to the structures of the two flavonoids (Fig. 1). Rutin is a glucoside of quercetin, incorporating the disaccharide rutinose. The presence of rutinose renders rutin less hydrophobic than quercetin and also more bulky. This latter fact may result in steric hindrance of its penetration of the hydrophobic pocket of Hb and may generally affect the orientation of the rutin molecules in relation to the protein. So the binding capacity of rutin with protein is less than that of quercetin. These results are agreed with the former studies of Bi et al. [18].
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Table 1 Binding and thermodynamic parameters of flavonoids–Hb interactions (a) quercetin and (b) rutin T (K)
Ksv (×104 L mol−1 )
K (×105 L mol−1 )
n
G (kJ mol−1 )
H (kJ mol−1 )
S (J mol−1 )
(a) Quercetin 298 308
5.34 4.71
6.02 2.51
1.28 1.19
−32.97 −31.84
−66.76
−113.39
(b) Rutin 298 308
5.12 2.34
2.34 0.87
1.16 1.05
−30.63 −29.14
−74.98
−149.83
3.3. Synchronous fluorescence and CD spectra To explore the structural change of Hb by addition of flavonoids, we measured synchronous fluorescence spectra (Fig. 4) of Hb with various amounts of flavonoids. The synchronous fluorescence spectra give information about the molecular environment in the vicinity of the chromosphere molecules. Yuan et al. [19] suggested a useful method to study the environment of amino acid residues by measuring the possible shift in wavelength emission maximum λmax , and the shift in position of emission maximum corresponding to the changes of the polarity around the chromophore molecule. When the D-value (λ) between excitation wavelength and emission wavelength are stabilized at 30 nm or 60 nm, the synchronous fluorescence gives the characteristic information of tyrosine residues or tryptophan residues [20]. The effects of flavonoids on Hb synchronous fluorescence spectroscopy are shown in Fig. 4 (date for quercetin not shown). It is apparent from Fig. 4 that the little stronger red shift of tryptophan residues exhibits fluorescence upon addition of rutin, whereas the emission maximum of tyrosine kept the position. The red shift of the emission maximum indicates that the conformation of Hb is changed and the polarity around the tryptophan residues is increased and the hydrophobicity is decreased.
Fig. 2. Fluorescence spectra of Hb in the prescence of (a) quercetin and (b) rutin at 25 ◦ C. Total concentrations of quercetin and rutin: (1) 0.0 mol L−1 ; (2) 0.5 × 10−5 mol L−1 ; (3)1.0 × 10−5 mol L−1 ; (4) 1.5 × 10−5 mol L−1 ; (5) 2.0 × 10−5 mol L−1 ; (6) 2.5 × 10−5 mol L−1 ; (7) 3.0 × 10−5 mol L−1 ; (8) 3.5 × 10−5 mol L−1 . The concentration of Hb is 3.0 × 10−6 mol L−1 .
Fig. 3. The plots of log[Q] vs. log((F0 − F)/F) at 25 ◦ C for (1) rutin and (2) quercetin. The concentration of Hb is 3.0 × 10−6 mol L−1 .
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Fig. 5. Synchronous fluorescence spectra of Hb in the prescence of rutin at 25 ◦ C: (a) λ = 30 nm; (b) λ = 60 nm. Total concentrations of rutin: (1) 0.0 mol L−1 ; (2) 1.0 × 10−5 mol L−1 ; (3) 1.5 × 10−5 mol L−1 ; (4) 2.0 × 10−5 mol L−1 ; (5) 2.5 × 10−5 mol L−1 ; (6) 3.0 × 10−5 mol L−1 ; (7) 3.5 × 10−5 mol L−1 . The concentration of Hb is 3.0 × 10−6 mol L−1 .
in the presence and absence of flavonoids are similar in shape, indicating that the structure of Hb after the addition of drug is also predominantly -helical. From these results, it is apparent that interaction of flavonoids with Hb causes a conformational change of the protein with the loss of helical stability. 3.4. Mode of binding There are essentially four types of noncovalent interactions that could play a role in ligand binding to proteins. These are hydrogen bonds, van der Waals forces, hydrophobic bonds, and electrostatic interactions [22]. If the enthalpy changes (H◦ ) do not vary significantly over the temperature range studied, then its value and that of S◦ can be determined from the Van’t Hoff equation: Fig. 4. Changes of the CD spectrum of Hb upon the addition of rutin at 298 K. Rutin to Hb (3.0 × 10−6 mol L−1 ) ratios: (1) 0:1, (2) 4:1, and (3) 8:1.
To gain a better understanding in physicochemical properties of flavonoid governing its spectral behavior and to draw relevant conclusions on the flavonoid–Hb binding mechanism, additional CD measurements were performed on flavonoid and the flavonoid–Hb complex. Take the binding of rutin molecule to Hb for example (data for quercetin not shown), Fig. 5 shows the CD spectra of the Hb and Hb–rutin complex obtained at pH 7.4. The CD spectra of Hb exhibited two negative minima at 209 nm and 221 nm, which is typical characterization of the helix structure of class proteins [21]. The binding of rutin to Hb decreases both of these bands, clearly indicating the decrease of the -helical content in protein. However, the CD spectra of Hb
ln K = −
S ◦ H ◦ + RT R
(3)
where K is the binding constant at the corresponding temperature and R is the gas constant. The equation provides the enthalpy (H◦ ) and entropy (S◦ ) changes on binding. The free energy of binding is estimated from the following relationship: G◦ = H ◦ − TS ◦
(4)
To obtain such information, the implications of the present results have been discussed in conjunction with thermodynamic characteristics obtained for flavonoids binding, and the thermodynamic parameters are listed in Table 1. The negative sign for G◦ indicates the spontaneity of the binding of flavonoids with Hb. Based on the characteristic signs of the thermodynamic
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parameters at the various interactions [23], the negative H◦ and S◦ values for flavonoids binding to Hb are associated with hydrogen bonding and van der Waals interaction. 3.5. The binding parameters on liposomal system 3.5.1. The distribution of flavonoids between the lecithin liposome and the aqueous phase UV spectra of flavonoids exhibit two major absorption bands in the region 200–400 nm. Band-I (300–380 nm) is considered to be associated with absorption due to the ring-B cinnamoyl system and band-II (200–280 nm) is considered to be associated with absorption due to the ring-A benzoyl system [24]. Fig. 6(a and b) shows the spectra of quercetin and rutin in water and lecithin liposomes at 25 ◦ C, respectively. In lecithin liposome, the absorbance at 340 nm increases and the band exhibits red shifts. The UV spectra of the flavonoids shift towards higher wavelength indicating that great numbers of flavonoid molecules are being incorporated into the liposomes. The flavonoid molecule has aromatic rings, its solubility in water is low and it exists in the anionic state with one or two charges. Therefore, quercetin and rutin will be easily extracted into the lecithin liposomes through hydrophobic interaction. Moreover, the lecithin molecule does carry a positive charge group due to the presence of N+ (CH3 )3 , it will definitely try to aggregate with flavonoid’s anion and this promotes the process of the location of flavonoids in liposome. The distribution coefficient KD of querctein and rutin between the lecithin liposome and the aqueous phase can be formulated as KD =
flavonoidL flavonoidW
(5)
Here flavonoidL and flavonoidW are the mole fractions of flavonoid in the lecithin liposome and aqueous phases, respectively. The distribution coefficient KD can be calculated by the following equation [25,26]: 1 1 1 1 × + = εΨ − ε W KD (εL − εW ) C εL − εW
(6)
Here C is the concentration of lecithin, ε the apparent molar absorptivity of flavonoids at given wavelength, εL and εm are the apparent molar absorptivities of flavonoids in the lecithin liposomes and aqueous phases, respectively. From the plot of 1/(ε − εW ) against 1/C, the distribution coefficient KD can be calculated from the slope and the intercept of the straight line (Fig. 7). The values of KD for quercetin and rutin are 1315.3 and 194.6, respectively. The distribution coefficient of quercetin is larger than that of rutin. This suggests the lecithin liposomes could incorporate more quercetin molecules. This result is probably concerned with the molecule structures of the two flavonoids. The presence of rutinose renders rutin molecule less hydrophobic than quercetin and also more bulky. These facts may result in steric hindrance of rutin incorporation into the hydrophobic region of liposome and may generally affect the concentrations of flavonoids in liposome.
Fig. 6. The absorption spectra of (a) quercetin and (b) rutin (3.0 × 10−5 mol L−1 ) in lecithin liposomes at 25 ◦ C. Lecithin concentration: (1) 0 g mL−1 , (2) 1.5 × 10−4 g mL−1 , and (3) 2.5 × 10−4 g mL−1 .
3.5.2. The effect of liposomes on the binding constants between the flavonoids and Hb Our former work shows that Hb can be adsorbed on lecithin liposomes by the hydrophobic interaction between Hb and lecithin molecules, which results in an increase of the microenvironmental polarity (I1 /I3 ) of pryene, a decrease of membrane fluidity (P), and microhydrophobicity of lecithin liposomes. With the increasing content of Hb, the permeability of the liposomal bilayer membranes increases [27]. These studies provide the foundation for the research of binding studied between flavonoids and protein in liposomes.
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Table 2 The effect of liposomes on the binding parameters between flavonoids and Hb at 289 K Compound
CHb (×10−6 mol L−1 )
CLecithin (×10−4 mol L−1 )
K (L mol−1 )
K /K
L Quercetin
3.0 3.0 1.0 1.0
1.0 2.5 1.0 2.5
7.19 × 104 5.01 × 104 1.65 × 105 1.14 × 105
0.12 0.08 0.27 0.19
Rutin
3.0 3.0 1.0 1.0
1.0 2.5 1.0 2.5
8.34 × 104 6.02 × 104 1.03 × 105 8.58 × 104
0.33 0.26 0.44 0.37
In the liposomal system, the fluorescence intensity of Hb also drops with the increase of flavonoids concentrations and the binding constants are determined at 25 ◦ C in the presence of lecithin liposome. The results shown in Table 2 reveal that the binding constants of the flavonoids with Hb are 8–44% of those without liposomes. This implies that when the two flavonoids are incorporated into liposome, they quench the fluorescence of the tryptophanyl residues of Hb to a lesser extent. In addition, the binding constant between quercetin and Hb (3.0 × 10−6 mol L−1 ) is reduced to 8% in liposomal system (2.5 × 10−4 g mol−1 ), but only to 26% for rutin in the same conditions. These results are due to the fact that the distribution coefficient of quercetin between the lecithin liposome and the aqueous phase is larger than that of rutin. A larger incorporated amount of quercetin into liposome induces the quench reaction between drug and protein to a lesser extent, so this difference in distribution coefficient of quercetin and rutin appears to have a significant effect on their binding affinity for Hb. Table 2 shows that the binding capacities of quercetin and rutin to Hb are greater at a higher liposome concentration (2.5 × 10−4 g mL−1 ) than those at lower liposome concentration
(1.0 × 10−4 g mL−1 ). This is due to the fact that the increase of the liposome concentration can bring more quercetin and rutin molecules entrapped into liposomal bilayer membranes, which can cause a lesser extent of the quenching reaction. From Table 2, it can also be seen that at higher Hb concentration (3.0 × 10−6 mol L−1 ), the binding constants of flavonoids with Hb are enhanced. These results are considered to be that the permeability of the liposomal bilayer membranes increases with the increasing content of Hb [27]. The increasing permeability of liposome at higher concentration of Hb enhances the amounts of quercetin and rutin molecules in aqueous phase, thus binding reactions between flavonoids and Hb become easier and the binding constants of quercetin and rutin to Hb are both increased. 4. Conclusions In this paper, the binding properties of two flavonoids (quercetin and rutin) to Hb were characterized by measuring the fluorescence and CD spectra. The results indicated a conformational change of Hb with addition of flavonoids. The binding parameters for flavonoids were obtained and the negative H◦ and S◦ values of thermodynamic parameters for flavonoids binding to Hb were associated with hydrogen bonding and van der Waals interaction. When incorporated into liposomes, quercetin and rutin could quench the fluorescence of the tryptophanyl residues of Hb to a lesser extent. However, the difference of the structure characteristics between them appears to have a significant effect on their incorporation amount in liopsome and the binding affinity for Hb. Acknowledgement This work is supported by National Natural Scientific Foundation of China (No. 20233010). References
Fig. 7. Plotting of (ε − εw)−1 vs. C−1 . The concentrations of (a) quercetin and (b) rutin are 3.0 × 10−5 mol L−1 .
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