Biomacromolecules 2004, 5, 1956-1961
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Probing the Binding of Scutellarin to Human Serum Albumin by Circular Dichroism, Fluorescence Spectroscopy, FTIR, and Molecular Modeling Method Jianniao Tian,† Jiaqin Liu,†,‡ Wenying He,† Zhide Hu,*,† Xiaojun Yao,† and Xingguo Chen† Department of Chemistry, Lanzhou University, Lanzhou 730000, China, and Mianyang Teacher’s College, Mianyang 621000, China Received June 7, 2004
The binding of scutellarin with human serum albumin (HSA) was investigated at four temperatures, 296, 303, 310, and 318 K, by fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR), and molecular modeling study at pH 7.40. The binding parameters were determined by Scatchard’s procedure, which are approximately consistent with the results of Stern-Volmer equation. The thermodynamic parameters were calculated according to the dependence of enthalpy change on the temperature as follows: ∆H° is a small negative value (-8.55 kJ/mol), whereas ∆S° is a positive value (65.15 J/mol K). Quenching of the fluorescence HSA in the presence of scutellarin was observed. Data obtained by fluorescence spectroscopy and CD experiment, FT-IR experiment, and molecular modeling method suggested that scutellarin can strongly bind to the HSA and the primary binding site of scutellarin is located in site I of HSA. It is considered that scutellarin binds to site I (subdomain II) mainly by a hydrophobic interaction and there are hydrogen bond interactions between the scutellarin and the residues Arg222 and Arg257. Introduction Serum albumin as one of most abundant carrier proteins plays an important role in the transport and disposition of endogenous and exogenous ligands present in blood.1 Distribution and metabolism of many biologically active compounds (drugs, natural products, etc.) in the body are correlated with their affinities toward serum albumin. Thus, the investigation of such molecules with respect to albumin binding is of imperative and fundamental importance. The molecular interactions between HSA and many compounds have been investigated successfully including many drugs.2-5 However, the binding of the components of natural plant medicine to proteins has seldom been investigated.6-7 The flavonoids comprise an important group of naturally occurring bioactive polyphenolics, ubiquitous in plants of higher genera.8,9 Recent interests on flavonoids have largely focused on their biological and relevant therapeutic applications. As far back as 1936, Szent-Gyorgyi10 first drew attention to the therapeutically beneficial role of dietary flavonoids. Currently, there is growing evidence for flavonoids with a wide range of therapeutic activities (against cancers, tumors, AIDs, allergies, inflammation, etc.) of high potency and low systemic toxicity.9,11 The antioxidative effects of flavonoids have been widely studied.12 Flavonoids such as quercetin, quercitrin, and rutin are found to suppress lipid peroxidation in spinach chloroplasts.13 It has recently been proved that serum albumin plays a decisive role in the * To whom correspondence should be addressed. Fax: +86-9318912582. E-mail:
[email protected]. † Lanzhou University. ‡ Mianyang Teacher’s College.
Scheme 1
transport and disposition of flavonoids.14 A number of biochemical and molecular biological investigations have revealed that proteins (including enzymes) are frequently the “targets” for therapeutically active flavonoids of both natural and synthetic origin.15 However, until today, very little was known about the mode of interactions of these compounds with their respective target proteins at the molecular level. Furthermore, the interaction of scutellarin with human serum albumin has not been reported. In this paper, the interaction of scutellarin (Huangqin Dai, Scheme 1, β-D-glucopyranosiduronic acid), which is the main active component of traditional Chinese medicine Scutellaria altissima L. and Baicalensis Georgi, with HSA was studied at pH 7.40 by spectroscopic methods including circular dichroism (CD), fluorescence spectroscopy, and Fourier transform infrared spectroscopy (FT-IR) spectra and molecular modeling study. The binding site of scutellarin was designated site I, the WF (warfarin) site on the HSA molecule, as indicated by the molecular modeling study of the interaction between HSA and scutellarin. The hydrophobic interaction was found to play a main role in the binding of this drug to HSA, but there are also a number of hydrogen bonds.
10.1021/bm049668m CCC: $27.50 © 2004 American Chemical Society Published on Web 08/18/2004
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Binding of Scutellarin to Human Serum Albumin
Materials and Experiments Human serum albumin (HSA, essentially fatty acid-free) was obtained from Sino-American Biotechnology Company. Scutellarin (analytical grade) was obtained from the National Institute for Control of Pharmaceutical and Products, China. 0.5 mol/L NaCl solution was used to keep the ion strength of solutions at 0.1 M. Tris-HCl buffer was selected to keep the pH of the solution at 7.40. HSA solution of 1.5 × 10-5 mol/L was prepared in pH 7.40 Tris-HCl buffer solution. A 1 mM scutellarin solution in ethanol was used in the binding experiments. All other chemicals were of analytical reagent grade. Binding Parameters. Fluorescence spectra were recorded using a Hitachi-850 spectrofluorophotometer (Japan) with a 150 W xenon lamp and a 1 cm quartz cell. The excitation and emission bandwidths were both 5 nm. The temperature of sample was kept by recycle water throughout experiment. The intrinsic fluorescence of HSA was obtained at 340 nm when excited at 280 nm. A quantitative analysis of the potential interaction between scutellarin and HSA was performed by fluoremetric titration. A 3 mL solution containing 1.5 × 10-6 mol/L HSA was titrated by successive additions of scutellarin solution (to give a final concentration of 3.0 × 10-5 mol/L), and the fluorescence intensity was measured (excitation at 280 nm and emission at 340 nm). All experiments were measured at four different temperatures (296, 303, 310, and 318 K). Using the fluorescence decrease, the association constants K for the complex of scutellarin with HSA at different temperatures were calculated. The binding parameters have been calculated using the Scatchard’s procedure.16 This method is based on the general equation r/Df ) nK - rK
(1)
where r is the moles of drug bound per mole of protein, Df is the molar concentration of free drug, n is binding site multiplicity per class of binding sites, and K is the equilibrium binding constant. Quenching data were also analyzed according to the Stern-Volmer equation17 RF0 1 1 1 ) + ∆RF [Q] fK f
(2)
where RF0 and ∆RF are the relative fluorescence intensities of protein in the absence and presence of quencher, respectively. f is the fractional maximum fluorescence intensity of protein summed up, and K is a constant. The dependence of RF0/∆RF on the reciprocal value of the quencher concentration 1/[Q] is linear with slope equal to the value of (fK)-1. The value 1/f is fixed on the ordinate. The association constant K is a quotient of an ordinate 1/f and slope (fK)-1. 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 ln K ) -∆H°/RT + ∆S°/R
(3)
In eq 3, K is the binding constant at corresponding temper-
ature and R is the gas constant. The temperatures used were 296, 303, 310, and 318 K. The enthalpy change (∆H°) is calculated from the slope of the van’t Hoff relationship. The free energy change is estimated from the following relationship: ∆G° ) ∆H° - T∆S°
(4)
CD and FT-IR Spectra. Circular dichroism (CD) measurements were made on a Jasco-20c automatic recording spectropolarimeter (Japan), using a 2 mm cell at 296K. The spectra were recorded in the range of 200-350 nm and the scan rate is 30 nm/min with a response time of 4 s. The induced ellipticity was the protein alone defined as the ellipticity of the drug-HSA mixture minus the ellipticity of drug alone at the same wavelength, and the results are expressed as molar ellipticity ([θ]) in deg cm2 dmol-1. The R-helical content of BSA was calculated from the [θ] value at 208 nm using the equation % helix ) {(-[θ]208 - 4000)/ (33000-4000)} × 100 as described by Lu et al.18 FI-IR measurements were carried out at room temperature on a Nicolet Nexus 670 FT-IR spectrometer (America) equipped with a Germanium attenuated total reflection (ATR) accessory, a DTGS KBr detector, and a KBr beam splitter. All spectra were taken via the attenuated total reflection (ATR) method with resolution of 4 cm-1 and 60 scans. Spectra processing procedures: spectra of buffer solution were collected at the same condition. Then, subtract the absorbance of buffer solution from the spectra of sample solution to get the FT-IR spectra of proteins. The subtraction criterion was that the original spectrum of protein solution between 2200 and 1800 cm-1 was a smooth straight.19 The ligand-protein solutions were prepared by mixing the ligand and protein solution to keep the final ligand-protein ratios at 0:1, 2:1 and 4:1. Molecular Modeling Study. The potential of the 3D structures of HSA was assigned according to the Amber 4.0 force field with Kollman-all-atom charges. The initial structures of all the molecules were generated by molecular modeling software Sybyl 6.9.20 The geometries of this drug were subsequently optimized using the Tripos force field with Gasteiger-Marsili charges. The AutoDock3.05 program21,22 was used to calculate the interaction modes between the drug and HSA. Lamarckian genetic algorithm (LGA) implemented in Autodock was applied to calculate the possible conformation of the drug that binds to the protein. During docking process, a maximum of 10 conformers was considered for the drug. The conformer with the lowest binding free energy was used for further analysis. All calculations were performed on Silicon Graphics Ocatane2 workstation. Results and Discussion Fluorescence Spectra. An intrinsic fluorescence study was performed to evaluate changes in tertiary structure caused by reaction of HSA with scutellarin. The effect of drug on HSA fluorescence intensity is shown in Figure 1. The fluorescence intensity of HSA was gradually decreased when the solution of scutellarin was added, which indicates scutellarin can bind to the HSA. The maximum wavelength
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Figure 1. Fluorescence spectra of the scutellarin-HSA system. The concentration of HSA was 1.5 × 10-6 mol/L, whereas the scutellarin concentration corresponds to 0, 0.6, 1.2, 1.8, and 2.4 × 10-5mol/L from the 0 to the 5(as the arrow indicates), 6 [scutellarin] ) 1.2 × 10-5 mol/L. T ) 296 K; pH 7.40; λex ) 280 nm, λem ) 340 nm.
Figure 2. Relative fluorescence intensity for the scutellarin-HSA interaction obtained by the titration with scutellarin. HSA concentration: 1.5 × 10-6 mol/L; pH 7.40; 1, 318 K; O, 310 K; b, 303 K; 9, 296 K; λex ) 280 nm, λem ) 340 nm.
of HSA shifted from 340 to 344 nm after the addition of scutellarin, so a slight red shift of the maximum emission wavelength was observed, and it could be deduced that conformational changes induced by the interaction lead to a further exposure of tryptophan residues to the polar solvent,23 which indicates that the binding site of scutellarin on HSA was adjacent to the sole tryptophan residue of HSA. The quantitative analysis of the binding of scutellarin to HSA was carried out using the fluorescence quenching at 340 nm at various temperatures as shown in Figure 2. With the increasing of the concentration of scutellarin the fluorescence intensity of system gradually decreased, and with the further addition of scutellarin, the fluorescence intensity of system decreased tardily in each titration curve which indicates the beginning of saturation of the HSA binding site. CD Spectra and FT-IR Spectra. To gain a better understanding in physicochemical properties of scutellarin governing its spectral behavior and to draw relevant conclusions on the scutellarin-HSA binding mechanism, additional
Tian et al.
Figure 3. CD spectra of the scutellarin-HSA complex at different drug to HSA ratios at pH 7.4 and 296 K. Drug to HSA (1.5 × 10-6 mol/L) ratios: 2, 0:1, b, 2:1, 9, 4:1.
CD and FI-IR spectroscopic measurements were performed on scutellarin and the scutellarin-HSA complex. Figure 3 shows the CD spectra of the HSA and HSA-scutellarin complex obtained at pH 7.40. The CD spectra of HSA exhibited two negative minima at 208 and 217 nm, which is typical characterization of the R-helix structure of class proteins.23 The interaction between scutellarin and HSA caused only a decrease in band intensity at all wavelengths of the far-UV CD without any significant shift of the peaks, indicating that this drug induces a slight decrease in the helic structure content of the protein. The content of R-helix decreased from 67.5% to 64.3% after the addition of scutellarin according to the quantitative analysis, which indicates that the interaction of scutellarin with HSA induced changes in the secondary structure of HSA. Infrared spectroscopy has long been used to a powerful method for investigating the secondary structures of proteins and their dynamics. In the IR region, the frequencies of bands due to the amide I, II, and III vibrations are sensitive to the secondary structure of proteins. Particularly, the amide I band is useful for the secondary structure studies. The protein amide I band 1600-1700 cm-1 (mainly CdO stretch) and amide II band 1548 cm-1 (C-N stretch coupled with N-H bending mode) both have a relationship with the secondary structure of protein. However, the amide I band is more sensitive to the change of protein secondary structure than amide II.24 Figure 4a showed the FT-IR spectrum of free HSA in Tris-HCl buffer, and the difference spectra of HSAscutellarin was also displayed in Figure 4b. The peak position of amide I moved from 1645 to 1656.58 cm-1, and amide II moved from 1550.5 to 1542.8 cm-1 in HSA infrared spectrum after interaction with scutellarin, which indicates that the secondary structure of HSA has been changed because of the interaction of scutellarin with HSA. This result was also in agreement with the result of CD experiment. Binding Parameters. Figures 5 and 6 are the Scatchard plot and the Stern-Volmer plots for the scutellarin-HSA system at different temperatures obtained from the fluoremetric titration, respectively. In Table 1, the binding constants obtained for the Scatchard and Stern-Volmer method are listed for scutellarin associated with HSA. The binding
Biomacromolecules, Vol. 5, No. 5, 2004 1959
Binding of Scutellarin to Human Serum Albumin Table 1. Thermodynamic Parameters of Scutellarin-HSA Interaction at pH 7.4 temp (K)
K (104M-1) (Stern-Volmer method)
K (104M-1) (Scatchard method)
n
∆G° (kJ/mol)
∆H° (kJ/mol)
∆S° (J/mol K)
296 303 310 318
8.32 7.58 6.95 6.41
7.80 7.49 6.97 6.43
1.22 1.22 1.20 1.23
-27.83 -28.29 -28.75 -29.27
-8.55
65.15
constants K of Stern-Volmer are likewise tabulated for comparison (Table 1) and approximate the former ones. The linearity of the Scatchard plots indicates that scutellarin binds to one class of sites on HSA, which was in full agreement with the number of binding site n, and the binding constants decreased with the increasing temperature. It shows that the binding between scutellarin and serum albumin is strong and the temperature has an effect on it. Thus, scutellarin can be stored and removed by protein in body. Binding Mode and Binding Site. 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.25,17 To obtain such information, the implications of the present results have been discussed in conjunction with
thermodynamic characteristics obtained for scutellarin binding, and the thermodynamic parameters were calculated from the Van’t Hoff plots. From Table 1, it can be seen that ∆H° is a small negative value (-8.55 kJ/mol), whereas ∆S° is a positive value (65.15 J/mol K). In these experiments, scutellarin-HSA complexes were accompanied by negative enthalpy changes (∆H°) and positive entropy changes (∆S°; Table 1), which indicates that the binding processes are entropically driven. The negative sign for ∆G° indicates the spontaneity of the binding of scutellarin with HSA. Based on the characteristic signs of the thermodynamic parameters at the various interactions,26 both positive enthalpy changes and entropy change values generally represent hydrophobic interactions. Moreover, a
Figure 5. Scatchard plot for the scutellarin-HSA at pH 7.40. HSA concentration: 1.5 × 10-6 mol/L; pH 7.40; 1, 318 K; O, 310 K; b, 303 K; 9, 296 K.
Figure 4. FT-IR spectra of free HSA (a) and difference spectra [(HSA solution + scutellarin solution) -/(scutellarin solution)] (b) in buffer solution in the region of 1800-1300 cm-1, [scutellarin] ) 6.0 × 10-5 mol/L, [HSA] ) 3.0 × 10-5 mol/L.
Figure 6. Stern-Volmer plot for scutellarin-HSA at pH 7.40. HSA concentration: 1.5 × 10-6mol/L; pH 7.40; O, 296 K; 9, 303 K; b, 310 K; 2, 318 K; λex ) 280 nm, λem ) 340 nm.
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Figure 7. Interaction mode between scutellarin and HSA, only residues around 8 Å of the ligand are displayed. The residues of HSA are represented using line and the ligand structure is represented using ball-and-stick model. The hydrogen bond between the ligand and the protein is represented using yellow dashed line. Table 2. Effect of Ionic Salt Strength on the Binding Constant of Scutellarin-HSA ionic salta strength (I)
K (104 M-1) (Scatchard method)
K (104 M-1) (Stern-Volmer method)
0.1 0.2 0.3
7.80 7.00 6.24
8.32 6.43 6.22
aIonic
salt: 1 M NaCl.
specific electrostatic interaction between ionic species in an aqueous solution is characterized by a positive ∆S° value and negative of small positive ∆H° value. For scutellarinHSA system, the main source of ∆G° value is derived from a large contribution of ∆S° term with little contribution from the ∆H° factor, so the main interaction is hydrophobic contact, but the electrostatic interaction cannot be excluded. This can be seen in Table 2. The ionic salt strength has an effect on the binding constant. With the increase of the ionic salt strength, the binding constant decreased. Therefore, the thermodynamic parameters for the interaction of scutellarin and HSA can be explained on the basis of plural intermolecular forces, such as hydrophobic and electrostatic interactions, rather than a single intermolecular force. Human serum albumin, a 585-residue protein, is monomeric but contains three structurally similar R-helical domains (I-III); each domain has two subdomains (A and B), which are six (A) and four (B) R-helices, respectively. Drugbinding sites I and II of HSA are located in hydrophobic cavities in subdomains IIA and IIIA, respectively. Several studies have shown that HSA is able to bind many ligands in several binding sites.27-28 There is a large hydrophobic
cavity present in subdomain IIA that many drugs can bind at. The crystal structure of HSA in complex with R-warfarin was taken from the Brookhaven Protein Data Bank (entry codes 1h9z).29 As shown in Figure 7, scutellarin binds within the subdomain IIA of the protein (The Warfarin Binding Pocket). The benzopyrone moiety is located within the binding pocket and the A and C rings are practically coplanar. It can be seen that site I is large enough to accommodate the scutellarin molecule. The interaction between drug and protein is dominated by hydrophobic contacts, but there are also a number of specific electrostatic interactions and a hydrogen bond. The phenyl moiety binds in a sub-pocket formed by the Lys-195, Trp-214, Ale-215, and Lys-199 amino acid residues. The scutellarin molecule binds in an apolar pocket with the 5-OH group and carboxyl group of the C ring, making a hydrogen bond with the several side chains of Arg222, the other hydrogen bond was formed between the mainchain carbonyl oxygen of Arg-257 and phenolic hydroxyl group. Conclusion The results of fluorescence quenching measurements and CD, FR-IR experiments, and molecular modeling study suggested that scutellarin could bind to HSA through the hydrophobic, electrostatic interaction and hydrogen bonds between scutellarin and Arg-222 and Arg-257 residues. In addition, the binding site is located in the hydrophobic pocket of subdomain IIA according to the molecular modeling study.
Binding of Scutellarin to Human Serum Albumin
References and Notes (1) Carter, D. C.; Ho, J. X. AdV. Protein Chem. 1994, 45, 153-203. (2) Norito, T.; Akihiko, H.; Hirofumi, K.; Michio, T.; Keishi, Y.; Ayaka, S.; Masaki, O. Pharm. Res. 1996, 13, 1015-1019. (3) Il’ichev, Y. V.; Jennifer, L. P.; John, D. S. J. Phys. Chem. B 2002, 106, 452-459. (4) Toshiaki, S.; Keishi, Y.; Tomoko, S.; Kragh-hansen, U.; Ayaka, S.; Masaki, O. Pharm. Res. 2001, 18, 520-524. (5) Fermin, M.; Jose, G. Chem-Biol. Interact. 1999, 121, 237-252. (6) Tian, J. N.; Liu, J. Q.; Zhang, J. Y.; Hu, Z. D.; Cheng, X. G. Chem. Pharm. Bull. 2003, 51, 579-582. (7) Liu, J. Q.; Tian, J. N.; Zhang, J. Y.; Hu, Z. D.; Cheng, X. G. Anal. Bioanal. Chem. 2003, 376, 864-867. (8) Mabry, T. J.; Markham, K. R.; Thomas, M. B. The systematic identification of flaVonoids; Springer-Verlag: New York, 1970. (9) Harborne, J. B. Flavonoids in the environment: structure-activity relationships. In Plant flaVonoids in biology and medicine. II. Biochemical, cellular, and medicinal properties; Cody, V., Middleton, E., Harborne, J. B., Beretz, A., Eds.; Alan R. Liss: New York, 1988; pp 17-27. (10) Rusznyak, S.; Szent-Gyorgyi, A. Nature 1936, 138, 27-27. (11) Lamson, S. W.; Brignall, M. S. Altern. Med. ReV. 2000, 5, 196208. (12) Takahama, J. J. Photochem. Photobiol. 1983, 38, 363-367. (13) Gschwendt, M.; Horn, F.; Kittstein, W.; Furstenberger, G.; Besemfekder, E.; Mark, F. Biochem. Biophys. Res. Commun. 1984, 124, 63-68. (14) Meijer, D. K. F.; Van der sluijs, P. Pharm. Res. 1989, 6, 105-118. (15) Sugio, S.; Kashima, A.; Mochizuki, S.; Noda, M.; Kobayashi, K. Protein Eng. 1999, 12, 439-446.
Biomacromolecules, Vol. 5, No. 5, 2004 1961 (16) Scatchard, G. Ann. N.Y. Acad. Sci. 1949, 51, 660-673. (17) Timaseff, S. N. Thermodynamics of protein interactions. In Proteins of biological fluids; Peeters, H., Ed.; Pergamon Press: Oxford, 1972; pp 511-519. (18) Lu, Z. X.; Cui, T.; Shi, Q. L. Applications of circular dichroism (CD) and optical rotatory dispersion (ORD) in molecular biology, 1st ed.; Science Press: Beijing, 1987. (19) Dong, A. C.; Huang, P.; Caughey, W. S. Biochemistry 1990, 29, 3303-3308. (20) SYBYL Software, Version 6.9, St. Louis, Tripos Associates Inc., 2002. (21) Morris, G. M.; Goodsell, D. S.; Huey, R.; Olson, A. J. J. Comput.Aided Mol. Des. 1996, 10, 293-304. (22) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.; Olson, A. J. J. Comput. Chem. 1998, 19, 16391662. (23) Gerbanowski, A.; Malabat, C.; Rabiller, C.; Gueguen, J. J. Agric. Food Chem. 1999, 47, 5218-5226. (24) Rahmelow, K.; Hubne, W. R. Anal. Biochem. 1996, 241, 5-10. (25) Klotz, I. M. Ann. N.Y. Acad. Sci. 1973, 226, 18-25. (26) Ross, P. D.; Subramanian, S. Biochemstry 1981, 20, 3096-3102. (27) He, X. M.; Carter, D. C. Nature 1992, 358, 209-215. (28) Curry, S.; Brick, P.; Franks, N. P. Biochim. Biophys. Acta 1999, 1441, 131-140. (29) Petitpas, I.; Bhattacharya, A. A.; Twine, S.; East, M.; Curry, S. J. Biol. Chem. 2001, 276, 22804-22809.
BM049668M
