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Effect of the glycosylation of flavonoids on interaction with protein
Spectrochimica Acta Part A 73 (2009) 972–975
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Short communication
Effect of the glycosylation of flavonoids on interaction with protein Hui Cao a,∗ , Donghui Wu a , Hongxian Wang a , Ming Xu b a b
School of Chemistry and Chemical Engineering, Nantong University, Nantong, Jiangsu 226007, PR China Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, NY 10021, USA
a r t i c l e
i n f o
Article history: Received 26 June 2008 Received in revised form 30 April 2009 Accepted 2 May 2009 Keywords: BSA Flavonoid Glycosylation Interaction Fluorescence quenching
a b s t r a c t In this paper, two flavonoid aglycones (baicalein, quercetin) and their glycosides (baicalin, quercitrin) were studied for their ability to bind protein by quenching the protein intrinsic fluorescence. From the spectra obtained, the bimolecular quenching constants, the apparent static binding constants, and binding sites values were calculated. The glycosylation of flavonoids decreases the binding affinity with protein. For quercetin and quercitrin, the binding constants for BSA were 3.65 × 107 and 6.47 × 103 L mol−1 , respectively. For baicalein and baicalin, the binding constants were 4.54 × 108 and 1.63 × 106 L mol−1 , respectively. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Flavonoids are the important phytonutrient components present in a wide range of fruits, vegetables, nuts and beverages, including wine and tea [1–6]. Flavonols are polyphenol compounds possessing two benzene rings joined by a linear three carbon chain (C2, C3, C4), represented as the C6–C3–C6 system. The results highlighted the importance of the flavonol moiety (2,3-double bond in conjugation with a 4-oxo group and a 3-hydroxyl group) and the 5,7-dihydroxylation at A-ring as important structural features for a significant in vitro antioxidant activity. Baicalin and baicalein are the most important bioactive components of Radix scutellariae [7,8]. Baicalin and baicalein exhibited wide range of pharmacological activities due to their antiallergic, antiinflammatory, antiatherogenic, antithrombotic, antibacterial, and antiviral properties. Quercetin (Fig. 1) is the most abundant bioflavonoid found in vegetable and fruits, and this compound is mainly present in the glycoside form, for example, as quercitrin. Quercetin, in addition to having antioxidant activity [9], quercetin has been suggested to inhibit neutrophil-mediated low-density lipoprotein oxidation [10] and influence GSH:GSSG ratios and protein thiolation in a tissue [11]. Quercitrin, the 3-O--glucoside of quercetin (Fig. 1), is a flavonol glycoside. Studies have demonstrated that when flavonols are present in the diet as aglycones, they could be partly absorbed
∗ Corresponding author. E-mail address: hui [email protected] (H. Cao). 1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2009.05.004
in the stomach, but their glycosidic forms cannot be absorbed [12]. There have been several studies on fluorescence quenching of proteins induced by flavonoids and other polyphenols [13–21]. However, the influence of glycosylation of flavonoids on binding characteristic with protein was not reported. In the present study, we evaluated the affinities of the flavonoid aglycones (baicalein, quercetin) and their glycosides (baicalin, quercitrin) with protein. 2. Material and methods 2.1. Apparatus Fluorescence spectra were recorded on a JASCO FP-6500 spectrofluorometer (Tokyo, Japan) equipped with a thermostated cell compartment. The UV–vis spectra were recorded on a UV-2450 spectrophotometer (Shimadzu, Japan). The pH measurements were carried out on a PHS-3C Exact Digital pH meter (Cole-Paemer Instrument Co.) equipped with Phonix Ag–AgCl reference electrode, which was calibrated with standard pH buffer solutions. 2.2. Reagents Bovine serum albumin (fraction V), quercitrin (≥98%), and baicalin (≥98%) were purchased from Sigma Co. (St. Louis, MO, USA). Baicalein (≥98%) and quercetin (≥98%) were obtained commercially from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). A working solution of flavonoid (1.0 × 10−4 mol L−1 ) was prepared by dissolving flavonoid in methanol–water solution (2:8, v/v). Tris–HCl buffer (0.20 mol L−1 ,
H. Cao et al. / Spectrochimica Acta Part A 73 (2009) 972–975
973
Fig. 1. Structures of flavonoids.
pH 7.4) containing 0.10 mol L−1 NaCl was selected to keep the pH value and maintain the ionic strength of the solution. The working solution of BSA (1.0 × 10−5 mol L−1 ) was prepared with tris–HCl buffer and stored in refrigerator prior to use. All other reagents and solvents were of analytical reagent grade and used without further purification unless otherwise noted. All aqueous solutions were prepared using newly double-distilled water. 2.3. Fluorescence and ultraviolet spectra Appropriate quantities of 1.0 × 10−4 mol L−1 flavonoid solution were transferred to a 10 mL flask, and then 1.0 mL of BSA solution was added and diluted to 10 mL with water. The resultant mixture was subsequently incubated at 310.15 K for 2 h. The solution was scanned on the fluorophotometer with the range of 290–500 nm. The fluorescent intensity at 340 nm was determined under the excitation at wavelength of 280 nm. The UV spectra were obtained by scanning the solution on the spectrophotometer with the wavelength range of 220–400 nm. The operations were carried out at room temperature. 2.4. Principles of fluorescence quenching Fluorescence quenching is described by the Stern–Volmer equation [16–20]: F0 = 1 + Kq 0 [Q ] = 1 + KSV [Q ] F
(1)
where F0 and F represent the fluorescence intensities in the absence and in the presence of quencher, Kq is the quenching rate constant of the bimolecular, KSV is the dynamic quenching constant, 0 is the average lifetime of the molecule without quencher and [Q] is the concentration of the quencher. Hence, Eq. (1) was applied to determine KSV by linear regression of a plot of F0 /F against [Q]. Fluorescence quenching could proceed via different mechanisms, usually classified as dynamic quenching and static quenching. Dynamic and static quenching can be distinguished by the quenching constant KSV . According to the literatures [18–22], for dynamic quenching, the maximum scatter collision
quenching constant of various quenchers with the biopolymer is 2.0 × 1010 L mol−1 s−1 . If the KSV is much greater than 2.0 × 1010 L mol−1 s−1 , it can be concluded that the quenching is not initiated by dynamic quenching, but probably partly by static quenching resulting from the formation of drug–BSA complex. For static quenching, the relationship between fluorescence quenching intensity and the concentration of quenchers can be described by modified form of the Stern–Volmer equation: lg
F0 − F = lg Ka + n lg [Q ] F
(2)
where Ka is the binding constant, and n is the number of binding sites per BSA. After the fluorescence quenching intensities on BSA at 340 nm were measured, the double-logarithm algorithm was assessed by Eq. (2). 3. Results and discussion 3.1. Effect of flavonols on BSA spectra The fluorescence spectra of BSA with the addition of baicalin (A), baicalein (B), quercitrin (C), and quercetin (D) were shown in Fig. 2. In all cases, the fluorescence intensity of BSA decreased remarkably with the increasing concentration of flavonoids. Weak blue shifts of the maximum em (1–2 nm) were observed for these four flavonoids. The dominant fluorophore is the indole group of tryptophan. Indole absorbs near 280 nm and emits near 340 nm. The emission of indole may be blue shifted if the group is buried within a native protein, and its emission may shift to longer wavelengths (red shift) when protein is unfolded [23]. When considering the effect of flavonols on the fluorescence spectra of BSA, there was weak blue shift of em . This suggests that there was some change in the immediate environment of the tryptophan residues, and the fact that the flavonoids were situated at close proximity to the tryptophan residue for the quenching to occur. This means that the molecular conformation of the protein was effected, which is agree to a recent study that has shown that the tertiary structure of proteins (human serum albumin, bovine serum
974
H. Cao et al. / Spectrochimica Acta Part A 73 (2009) 972–975
Fig. 2. The quenching effect of flavonoids on BSA fluorescence intensity. ex = 280 nm; BSA, 1.00 × 10−6 mol L−1 ; A, B, and C, a–k: 1.00, 2.00, 3.00, 4.00, 5.00, 6.00, 7.00, 8.00, 9.00, 10.00 (×10−6 mol L−1 ) of baicalin (A), baicalein (B), and quercitrin (C); D a–k: 0.00, 2.00, 4.00, 6.00, 8.00, 10.00, 12.00, 14.00, 16.00, 18.00, 20.00 (×10−7 mol L−1 ) of quercetin (D).
albumin, soy glycinin, and lysozyme) changes upon binding of phenolic compounds (chlorogenic, ferulic, and gallic acids, quercetin, rutin, and isoquercetin), while the secondary structure remains intact.
Fig. 3. Tryptophan fluorescence quenching of BSA (1.00 × 10−6 mol L−1 ) plotted as extinction of BSA tryptophans (F/F0 , %) against flavonoid concentration for baicalin (a), baicalein (b), and quercitrin (c), and quercetin (d) at 310.15 K. The fluorescence emission intensity was recorded at ex = 280 nm and em = 340 nm.
3.2. Fluorescence quenching of BSA The raw data for quenching of BSA fluorescence by addition of baicalin (a), baicalein (b), quercitrin (c), and quercetin (d) were shown in Fig. 3. These results indicate that the changes of the environment of tryptophan residues depend on the flavonoid structure. The experiment with baicalin was found to lead to 90% quenching, while quercitrin quenched only 30%. Baicalein and quercetin quenched 50–60% of BSA fluorescence. The extinction of BSA tryptophans by quercetin decreased rapidly, but extinction by others decreased slowly. Fig. 4 shows the Stern–Volmer plots for the BSA fluorescence quenching by baicalin (a), baicalein (b), quercitrin (c), and quercetin (d). In our research, both dynamic and static quenching were involved for baicalin (a), baicalein (b), quercitrin (c), and quercetin (d), which was demonstrated by the fact that the Stern–Volmer plot slightly deviated from linearity toward the y-axis at high flavonol concentrations. In the linear range of Stern–Volmer regression curve the average quenching constants for, baicalin, baicalein, quercitrin, and quercetin at 310.15 K were 2.30 × 105 (R = 0.9929), 9.36 × 105 (R = 0.9998), 4.86 × 104 (R = 0.9999), and 6.04 × 105 (R = 0.9945) L mol−1 , respectively. Because the fluorescence life time of the biopolymer is 10−8 s, the quenching constants Kq for baicalin, baicalein, quercitrin, and quercetin were calculated to be 2.30 × 1013 , 9.36 × 1013 , 4.86 × 1012 , and
H. Cao et al. / Spectrochimica Acta Part A 73 (2009) 972–975
975
Table 1 The binding parameters for the system of flavonoids-BSA (310.15 K). Flavonols
lg Ka
n
R
Quercitrin Quercetin Baicalein Baicalin
3.8107 7.5626 8.6571 6.2128
0.814 1.291 1.421 1.173
0.9835 0.9946 0.9951 0.9961
concentration and the metabolism of drug in the blood stream. Thus, the drug–albumin complex may be considered as a model for gaining fundamental insights into drug–protein interactions. 3.3. Binding constant and binding sites
Fig. 4. The Stem–Volmer curves of fluorescence quenching of BSA by baicalin (a), baicalein (b), and quercitrin (c), and quercetin (d) at 310.15 K.
Fig. 5 shows the double-logarithm curve and Table 1 give the corresponding calculated results. The apparent binding constants (Ka ) and the binding sites values (n) between flavonols and BSA decreased after glycosylation. After glycoside substituted on flavonoids, the steric hindrance may take place, which weakened the binding affinity. References
Fig. 5. Double-log plot of baicalin (a), baicalein (b), and quercitrin (c), and quercetin (d) quenching effect on BSA fluorescence at 310.15 K.
6.04 × 1013 L mol−1 L mol−1 s−1 , respectively. Considering that in our experiment the rate constants of the protein quenching procedure initiated by flavonoids were 102 –103 -fold higher than the maximum value possible for diffusion limited quenching in solution (∼1010 L mol−1 s−1 ), which suggests that the quenching is not initiated by dynamic collision and that there is a specific interaction occurring between BSA and flavonols. The drug–protein interaction may result in the formation of a stable protein–drug complex, which has important effect on the distribution, free
[1] T.F. Jiang, Y.H. Wang, Z.H. Lv, M.E. Yue, J. Pharmaceut. Biomed. 43 (2007) 854. [2] P. García-Macías, M. Ordidge, E. Vysini, S. Waroonphan, N.H. Battey, M.H. Gordon, P. Hadley, P. John, J.A. Lovegrove, A. Wagstaffe, J. Agric. Food Chem. 55 (2007) 10168. [3] M. Leopoldini, N. Russo, M. Toscano, J. Agric. Food Chem. 55 (2007) 7944. [4] L.Z. Lin, S. Lu, J.M. Harnly, J. Agric. Food Chem. 55 (2007) 1321. [5] H. Qu, Y. Ma, K. Yu, Y. Cheng, J. Pharmaceut. Biomed. 43 (2007) 66. [6] H.D. Chludil, G.B. Corbino, S.R. Leicach, J. Agric. Food Chem. 56 (2008) 5050. [7] K. Yu, Y. Gong, Z. Lin, Y. Cheng, J. Pharmaceut. Biomed. 43 (2007) 540. [8] D.S. Chou, J.J. Lee, G. Hsiao, C.Y. Hsieh, Y.J. Tsai, T.F. Chen, J.R. Sheu, J. Agric. Food Chem. 55 (2007) 649. [9] K.L. Wolfe, R.H. Liu, J. Agric. Food Chem. 56 (2008) 8404. [10] W.M. Loke, J.M. Proudfoot, A.J. Mckinley, P.W. Needs, P.A. Kroon, J.M. Hodgson, K.D. Croft, J. Agric. Food Chem. 56 (2008) 3609. [11] K.J. Meyers, J.L. Rudolf, A.E. Mitchell, J. Agric. Food Chem. 56 (2008) 830. [12] V. Crespy, C. Morand, C. Besson, C. Manach, C. Demigne, C. Remesy, J. Agric. Food Chem. 50 (2002) 618. [13] Y.Q. Wang, H.M. Zhang, G.C. Zhang, W.H. Tao, Z.H. Fei, Z.T. Liu, J. Pharmaceut. Biomed. 43 (2007) 1869. [14] J. Xiao, X. Chen, L. Zhang, S.G. Talbot, G.C. Li, M. Xu, J. Agric. Food Chem. 56 (2008) 910. [15] J.B. Xiao, M. Suzuki, X.Y. Jiang, X.Q. Chen, K. Yamamoto, M. Xu, J. Agric. Food Chem. 56 (2008) 2350. [16] L.H. Riihimaki, M.J. Vainio, J.M.S. Heikura, K.H. Valkonen, V.T. Virtanen, P.M. Vuorela, J. Agric. Food Chem. 56 (2008) 7721. [17] S. Soares, N. Mateus, V. Freitas, J. Agric. Food Chem. 55 (2007) 6726. [18] J.B. Xiao, J.W. Chen, H. Cao, F.L. Ren, C.S. Yang, Y. Chen, M. Xu, J. Photochem. Photobiol. A 191 (2007) 222. [19] J.B. Xiao, J. Shi, H. Cao, S.D. Wu, F.L. Ren, M. Xu, J. Pharmaceut. Biomed. 45 (2007) 609. [20] J.B. Xiao, X.Q. Chen, X.Y. Jiang, M. Hilczer, M. Tachiya, J. Fluoresc. 18 (2008) 671. [21] Y.Z. Zhang, B. Zhou, Y.X. Liu, C.X. Zhou, X.L. Ding, Y. Liu, J. Fluoresc. 18 (2008) 109. [22] Y. He, Y.W. Wang, L.F. Tang, H. Liu, W. Chen, Z.L. Zheng, G.L. Zou, J. Fluoresc. 18 (2008) 433. [23] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed., Kluwer, New York, 1999, p. 237.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Short communication
Effect of the glycosylation of flavonoids on interaction with protein Hui Cao a,∗ , Donghui Wu a , Hongxian Wang a , Ming Xu b a b
School of Chemistry and Chemical Engineering, Nantong University, Nantong, Jiangsu 226007, PR China Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, NY 10021, USA
a r t i c l e
i n f o
Article history: Received 26 June 2008 Received in revised form 30 April 2009 Accepted 2 May 2009 Keywords: BSA Flavonoid Glycosylation Interaction Fluorescence quenching
a b s t r a c t In this paper, two flavonoid aglycones (baicalein, quercetin) and their glycosides (baicalin, quercitrin) were studied for their ability to bind protein by quenching the protein intrinsic fluorescence. From the spectra obtained, the bimolecular quenching constants, the apparent static binding constants, and binding sites values were calculated. The glycosylation of flavonoids decreases the binding affinity with protein. For quercetin and quercitrin, the binding constants for BSA were 3.65 × 107 and 6.47 × 103 L mol−1 , respectively. For baicalein and baicalin, the binding constants were 4.54 × 108 and 1.63 × 106 L mol−1 , respectively. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Flavonoids are the important phytonutrient components present in a wide range of fruits, vegetables, nuts and beverages, including wine and tea [1–6]. Flavonols are polyphenol compounds possessing two benzene rings joined by a linear three carbon chain (C2, C3, C4), represented as the C6–C3–C6 system. The results highlighted the importance of the flavonol moiety (2,3-double bond in conjugation with a 4-oxo group and a 3-hydroxyl group) and the 5,7-dihydroxylation at A-ring as important structural features for a significant in vitro antioxidant activity. Baicalin and baicalein are the most important bioactive components of Radix scutellariae [7,8]. Baicalin and baicalein exhibited wide range of pharmacological activities due to their antiallergic, antiinflammatory, antiatherogenic, antithrombotic, antibacterial, and antiviral properties. Quercetin (Fig. 1) is the most abundant bioflavonoid found in vegetable and fruits, and this compound is mainly present in the glycoside form, for example, as quercitrin. Quercetin, in addition to having antioxidant activity [9], quercetin has been suggested to inhibit neutrophil-mediated low-density lipoprotein oxidation [10] and influence GSH:GSSG ratios and protein thiolation in a tissue [11]. Quercitrin, the 3-O--glucoside of quercetin (Fig. 1), is a flavonol glycoside. Studies have demonstrated that when flavonols are present in the diet as aglycones, they could be partly absorbed
∗ Corresponding author. E-mail address: hui [email protected] (H. Cao). 1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2009.05.004
in the stomach, but their glycosidic forms cannot be absorbed [12]. There have been several studies on fluorescence quenching of proteins induced by flavonoids and other polyphenols [13–21]. However, the influence of glycosylation of flavonoids on binding characteristic with protein was not reported. In the present study, we evaluated the affinities of the flavonoid aglycones (baicalein, quercetin) and their glycosides (baicalin, quercitrin) with protein. 2. Material and methods 2.1. Apparatus Fluorescence spectra were recorded on a JASCO FP-6500 spectrofluorometer (Tokyo, Japan) equipped with a thermostated cell compartment. The UV–vis spectra were recorded on a UV-2450 spectrophotometer (Shimadzu, Japan). The pH measurements were carried out on a PHS-3C Exact Digital pH meter (Cole-Paemer Instrument Co.) equipped with Phonix Ag–AgCl reference electrode, which was calibrated with standard pH buffer solutions. 2.2. Reagents Bovine serum albumin (fraction V), quercitrin (≥98%), and baicalin (≥98%) were purchased from Sigma Co. (St. Louis, MO, USA). Baicalein (≥98%) and quercetin (≥98%) were obtained commercially from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). A working solution of flavonoid (1.0 × 10−4 mol L−1 ) was prepared by dissolving flavonoid in methanol–water solution (2:8, v/v). Tris–HCl buffer (0.20 mol L−1 ,
H. Cao et al. / Spectrochimica Acta Part A 73 (2009) 972–975
973
Fig. 1. Structures of flavonoids.
pH 7.4) containing 0.10 mol L−1 NaCl was selected to keep the pH value and maintain the ionic strength of the solution. The working solution of BSA (1.0 × 10−5 mol L−1 ) was prepared with tris–HCl buffer and stored in refrigerator prior to use. All other reagents and solvents were of analytical reagent grade and used without further purification unless otherwise noted. All aqueous solutions were prepared using newly double-distilled water. 2.3. Fluorescence and ultraviolet spectra Appropriate quantities of 1.0 × 10−4 mol L−1 flavonoid solution were transferred to a 10 mL flask, and then 1.0 mL of BSA solution was added and diluted to 10 mL with water. The resultant mixture was subsequently incubated at 310.15 K for 2 h. The solution was scanned on the fluorophotometer with the range of 290–500 nm. The fluorescent intensity at 340 nm was determined under the excitation at wavelength of 280 nm. The UV spectra were obtained by scanning the solution on the spectrophotometer with the wavelength range of 220–400 nm. The operations were carried out at room temperature. 2.4. Principles of fluorescence quenching Fluorescence quenching is described by the Stern–Volmer equation [16–20]: F0 = 1 + Kq 0 [Q ] = 1 + KSV [Q ] F
(1)
where F0 and F represent the fluorescence intensities in the absence and in the presence of quencher, Kq is the quenching rate constant of the bimolecular, KSV is the dynamic quenching constant, 0 is the average lifetime of the molecule without quencher and [Q] is the concentration of the quencher. Hence, Eq. (1) was applied to determine KSV by linear regression of a plot of F0 /F against [Q]. Fluorescence quenching could proceed via different mechanisms, usually classified as dynamic quenching and static quenching. Dynamic and static quenching can be distinguished by the quenching constant KSV . According to the literatures [18–22], for dynamic quenching, the maximum scatter collision
quenching constant of various quenchers with the biopolymer is 2.0 × 1010 L mol−1 s−1 . If the KSV is much greater than 2.0 × 1010 L mol−1 s−1 , it can be concluded that the quenching is not initiated by dynamic quenching, but probably partly by static quenching resulting from the formation of drug–BSA complex. For static quenching, the relationship between fluorescence quenching intensity and the concentration of quenchers can be described by modified form of the Stern–Volmer equation: lg
F0 − F = lg Ka + n lg [Q ] F
(2)
where Ka is the binding constant, and n is the number of binding sites per BSA. After the fluorescence quenching intensities on BSA at 340 nm were measured, the double-logarithm algorithm was assessed by Eq. (2). 3. Results and discussion 3.1. Effect of flavonols on BSA spectra The fluorescence spectra of BSA with the addition of baicalin (A), baicalein (B), quercitrin (C), and quercetin (D) were shown in Fig. 2. In all cases, the fluorescence intensity of BSA decreased remarkably with the increasing concentration of flavonoids. Weak blue shifts of the maximum em (1–2 nm) were observed for these four flavonoids. The dominant fluorophore is the indole group of tryptophan. Indole absorbs near 280 nm and emits near 340 nm. The emission of indole may be blue shifted if the group is buried within a native protein, and its emission may shift to longer wavelengths (red shift) when protein is unfolded [23]. When considering the effect of flavonols on the fluorescence spectra of BSA, there was weak blue shift of em . This suggests that there was some change in the immediate environment of the tryptophan residues, and the fact that the flavonoids were situated at close proximity to the tryptophan residue for the quenching to occur. This means that the molecular conformation of the protein was effected, which is agree to a recent study that has shown that the tertiary structure of proteins (human serum albumin, bovine serum
974
H. Cao et al. / Spectrochimica Acta Part A 73 (2009) 972–975
Fig. 2. The quenching effect of flavonoids on BSA fluorescence intensity. ex = 280 nm; BSA, 1.00 × 10−6 mol L−1 ; A, B, and C, a–k: 1.00, 2.00, 3.00, 4.00, 5.00, 6.00, 7.00, 8.00, 9.00, 10.00 (×10−6 mol L−1 ) of baicalin (A), baicalein (B), and quercitrin (C); D a–k: 0.00, 2.00, 4.00, 6.00, 8.00, 10.00, 12.00, 14.00, 16.00, 18.00, 20.00 (×10−7 mol L−1 ) of quercetin (D).
albumin, soy glycinin, and lysozyme) changes upon binding of phenolic compounds (chlorogenic, ferulic, and gallic acids, quercetin, rutin, and isoquercetin), while the secondary structure remains intact.
Fig. 3. Tryptophan fluorescence quenching of BSA (1.00 × 10−6 mol L−1 ) plotted as extinction of BSA tryptophans (F/F0 , %) against flavonoid concentration for baicalin (a), baicalein (b), and quercitrin (c), and quercetin (d) at 310.15 K. The fluorescence emission intensity was recorded at ex = 280 nm and em = 340 nm.
3.2. Fluorescence quenching of BSA The raw data for quenching of BSA fluorescence by addition of baicalin (a), baicalein (b), quercitrin (c), and quercetin (d) were shown in Fig. 3. These results indicate that the changes of the environment of tryptophan residues depend on the flavonoid structure. The experiment with baicalin was found to lead to 90% quenching, while quercitrin quenched only 30%. Baicalein and quercetin quenched 50–60% of BSA fluorescence. The extinction of BSA tryptophans by quercetin decreased rapidly, but extinction by others decreased slowly. Fig. 4 shows the Stern–Volmer plots for the BSA fluorescence quenching by baicalin (a), baicalein (b), quercitrin (c), and quercetin (d). In our research, both dynamic and static quenching were involved for baicalin (a), baicalein (b), quercitrin (c), and quercetin (d), which was demonstrated by the fact that the Stern–Volmer plot slightly deviated from linearity toward the y-axis at high flavonol concentrations. In the linear range of Stern–Volmer regression curve the average quenching constants for, baicalin, baicalein, quercitrin, and quercetin at 310.15 K were 2.30 × 105 (R = 0.9929), 9.36 × 105 (R = 0.9998), 4.86 × 104 (R = 0.9999), and 6.04 × 105 (R = 0.9945) L mol−1 , respectively. Because the fluorescence life time of the biopolymer is 10−8 s, the quenching constants Kq for baicalin, baicalein, quercitrin, and quercetin were calculated to be 2.30 × 1013 , 9.36 × 1013 , 4.86 × 1012 , and
H. Cao et al. / Spectrochimica Acta Part A 73 (2009) 972–975
975
Table 1 The binding parameters for the system of flavonoids-BSA (310.15 K). Flavonols
lg Ka
n
R
Quercitrin Quercetin Baicalein Baicalin
3.8107 7.5626 8.6571 6.2128
0.814 1.291 1.421 1.173
0.9835 0.9946 0.9951 0.9961
concentration and the metabolism of drug in the blood stream. Thus, the drug–albumin complex may be considered as a model for gaining fundamental insights into drug–protein interactions. 3.3. Binding constant and binding sites
Fig. 4. The Stem–Volmer curves of fluorescence quenching of BSA by baicalin (a), baicalein (b), and quercitrin (c), and quercetin (d) at 310.15 K.
Fig. 5 shows the double-logarithm curve and Table 1 give the corresponding calculated results. The apparent binding constants (Ka ) and the binding sites values (n) between flavonols and BSA decreased after glycosylation. After glycoside substituted on flavonoids, the steric hindrance may take place, which weakened the binding affinity. References
Fig. 5. Double-log plot of baicalin (a), baicalein (b), and quercitrin (c), and quercetin (d) quenching effect on BSA fluorescence at 310.15 K.
6.04 × 1013 L mol−1 L mol−1 s−1 , respectively. Considering that in our experiment the rate constants of the protein quenching procedure initiated by flavonoids were 102 –103 -fold higher than the maximum value possible for diffusion limited quenching in solution (∼1010 L mol−1 s−1 ), which suggests that the quenching is not initiated by dynamic collision and that there is a specific interaction occurring between BSA and flavonols. The drug–protein interaction may result in the formation of a stable protein–drug complex, which has important effect on the distribution, free
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