European Journal of Medicinal Chemistry 93 (2015) 492e500
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Comparison between loureirin A and cochinchinenin C on the interaction with human serum albumin Xu Chen a, 1, Kai Qian b, 1, Qin Chen b, * a b
Experimental Center for Life Scinence, Shanghai University, Shanghai, PR China Shanghai Key Lab Bioenergy Crops, School of Life Sciences, Shanghai University, Shanghai, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 April 2014 Received in revised form 16 February 2015 Accepted 17 February 2015 Available online 24 February 2015
The interactions of loureirin A (LA) and cochinchinenin C (CC) with human serum albumin (HSA) under simulated physiological conditions (pH ¼ 7.4) have been studied with fluorescence, UVevis absorption spectroscopic method and molecular docking technique. The results indicated that there was a synergistic interaction between LA and CC, and the fluorescence quenching of HSA by LA (or CC) was a combined quenching procedure (dynamic and static quenching). At low compound concentrations, the quenching constants KSV of CC was larger than that of LA, which meant the CC efficacy may be better than that of LA. The negative △H and △S values suggested hydrogen bonds and van der Waals forces played the major role in the binding of LA (or CC) to HSA. The efficiency of energy transfer and distance between the compounds and HSA was calculated. Moreover, the results of synchronous and three-dimensional fluorescence demonstrated that the HSA microenvironment was changed in the presence of LA (or CC). Finally, the binding of LA (or CC) to HSA was modeled by molecular docking, which is in good accordance with the experimental studies. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Loureirin A Cochinchinenin C Human serum albumin Fluorescence quenching Molecular docking
1. Introduction Loureirin A (LA) and cochinchinenin C (CC) (structures shown in Fig. 1), which belong to flavonoids, are the important bioactive constituents of Dragon's blood [1]. As is well-known, Dragon's blood is one of the renowned traditional medicines, of which from Dranaena cochinchinensis (Lour). S.C. Chen is named ‘Resina Draaconis’ in China. Dragon's blood has been widely used for diverse biological applications including antithrombotic, anti-blood stasis, anticoagulant, analgesic, blood circulation promotion, reducing high level of blood sugar and other important therapeutic activities [2e7]. And there are different affinity and activity between LA and CC, which can be attributed to their different molecular structures and different molecular weights. In previous studies, LA was reported to exhibit analgesic activity [1], and CC was reported with anti-Helicobacter pylori and thrombin inhibitory activities [8]. Human serum albumin (HSA) is the most abundant protein in plasma and has many physiological functions. The principle function of HSA is to act as a carrier for many endogenous and
* Corresponding author. E-mail address: [email protected] (Q. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2015.02.025 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
exogenous molecules and metabolites to particular biological targets [9]. Therefore, HSA plays an important role in the distribution, metabolism, efficacy and interaction with the drug target tissues [10]. The drug pharmaceutical activity level depends on the interaction with targeting molecules, mode of action and affinity size, while the structural differences in drug often lead to changes in its biological activity [11,12]. Thus, the investigation of drugs binding to HSA is of important significance [13]. In the present work, a comparative study of the interaction between HSA and LA (or CC) was undertaken by fluorescence and UVevis absorption spectroscopic and docking simulation methods. The spectroscopic techniques, which are sensitive and relatively simple, are often used to monitor the molecular interactions [14,15]. The results obtained from these methods not only revealed the LA (or CC) and HSA interaction mechanism, but also may provide some valuable information for drug design and pharmaceutical research with comparative studies on LA and CC interactions with HSA. 2. Experimental method 2.1. Materials Human serum albumin (approximately 99% and fatty acid free)
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Fig. 1. Molecular structures of loureirin A (A) and cochinchinenin C (B).
was purchased from Sigma Chemical Company (USA), loureirin A and cochinchinenin C were purchased from National Institute for the Control of Pharmaceutical and Biological Products (China). The stock solution of human serum albumin was prepared in 0.1 M TriseHCl buffer with pH ¼ 7.4 containing 0.1 M sodium chloride. A 1.5 106 M working solution of HSA was prepared by appropriate dilution of the stock solution. Loureirin A was directly dissolved in anhydrous alcohol as stock solution (2.0 103 M). Stock solution of cochinchinenin C (0.4 103 M) was also prepared by dissolving appropriate amount of the drugs in anhydrous alcohol. All other reagents were of guarantee grade and double distilled water was used throughout. 2.2. Apparatus All fluorescence measurements were performed on a Spectrofluorimeter Model LS-55 (PerkinElmer, USA) equipped with a quartz cell (10-mm) and a thermostat bath. The absorption spectra were recorded on Cary100 UVevis spectrophotometer (Varian, Australia) with a 10-mm quartz cuvette at room temperature. The pH values were measured on a pHS-3C Digital pH-meter (Shanghai Lei Ci device Works, China).
were obtained upon excitation wavelength range of 200e480 nm with 10 nm increments and monitoring the emission spectra from 220 to 500 nm. 2.3.2. UVevis absorption spectra The absorption titration were carried out by keeping the concentration of HSA in 1.5 106 M, while the LA (or CC) concentration was varied from 0 to 18 106 M. Changes in absorbance at 280 nm are convincing evidences for the formation of compoundeHSA complex. The absorption spectra of LA and CC (1.5 106 M) were also recorded. 2.3.3. Molecular docking The crystal structure of the HSA (PDB ID: 1h9z) was obtained from the protein data bank. Water molecules were removed, and hydrogen atoms were added. The structure of LA and CC were created as mol files using ChemDraw Ultra (Ver. 9.0). All simulations were performed by using Discovery Studio 4.0 software, which is an interactive molecular graphics program assisting the understanding of the compoundeprotein interaction. 3. Results and discussion
2.3. Procedures
3.1. Fluorescence quenching of HSA
2.3.1. Fluorescence spectra 3.0 mL solution, containing 1.5 106 M HSA, was titrated by successive additions of 1.0 106 L LA (or CC) stock solution. The solution was allowed to stand for 5 min to equilibrate. Fluorescence emission spectra were recorded from 300 to 450 nm with an excitation wavelength at 280 nm at three different temperatures (300, 305 and 310 K). On the other hand, the D-value between excitation and emission wavelengths (Dl) was set at 15 and 60 nm for the synchronous fluorescence spectra measurements, respectively. Excitation wavelength was set from 270 to 320 nm. Both excitation and emission bandwidths were set at 10 nm. Three-dimensional (3-D) fluorescence spectra of HSA (1.5 106 M) and the compoundHSA complexes (2:1 M ratios)
The fluorescence emission spectra of HSA in the presence of different LA and CC concentrations are recorded at three temperatures (300, 305 and 310 K) and one of them (310 K) are shown in Fig. 2. The excitation wavelength was set at 280 nm, so that both tyrosyl and tryptophyl fluorophores were excited [16]. Fig. 2 suggests that HSA exhibits a stronger fluorescence peak, while LA and CC display weaker intrinsic fluorescence under the same experimental conditions. Furthermore, the fluorescence intensity of HSA drops gradually with the increase of LA concentration, along with a slight red shift (347.5 / 348.5 nm). With the increase of CC concentration, binding sites on HSA are increasingly saturated, which can be explained by the presence of aggregates of CC. The resulting steric hindrance may take place that weaken the affinity of CC to
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Fig. 2. Emission spectra of HSA with LA (A) and CC (B). CHSA ¼ 1.5 106 M, C LA, a / h: 0, 0.65, 1.3, 1.95, 2.6, 3.25, 3.9 and 4.55 106 M; C CC, a / h: 0, 0.14, 0.28, 0.42, 0.56, 0.7, 0.84, and 0.98 106 M; T ¼ 310 K.
HSA. As a result, the fluorescence intensity of HSA is almost no longer reduced and the position of the emission maximum wavelength shows a moderate blue shift (346.5 / 344 nm). Therefore, the observation indicates that the intrinsic fluorescence of HSA is quenched accompanying with a microenvironmental change around HSA after the addition of LA (or CC). Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interactions with a quencher molecule, such as excited-state reactions, molecular rearrangement, energy transfer, groundestate complex formation, and collisional quenching. Quenching can be induced by dynamic and static processes, which are caused by collisional encountering between the fluorophore and quencher and non-fluorescent ground state complex formation, respectively. However, in many instances, the fluorophore can be quenched both by collisions and by complex formation with the same quencher. The characteristic feature of the SterneVolmer plot exhibits an upward curvature for the distinct situation, concave towards the y axis at high quencher concentration [17]. To confirm the quenching mechanism, the fluorescence quenching of HSA at different temperatures (300, 305 and 310 K) has been analyzed by SterneVolmer equation:
F0 =F ¼ 1 þ kq t0 ½Q ¼ 1 þ Ksv Q
(1)
where F0 and F represent the fluorescence intensities of HSA in absence and presence of quencher, respectively, [Q] is the quencher concentration, kq is bimolecular quenching rate constant, Ksv is the SterneVolmer quenching constant and t0 is the average lifetime of the biomolecule without quencher (t0 ¼ 108 s) [18,19]. Fig. 3 displays the SterneVolmer plots for the fluorescence quenching of HSA by LA and CC at different temperatures. In Fig. 3A, the upward curves at high concentrations suggest that the fluorescence quenching mechanism of LA with HSA is a combined one. The plateau at high concentrations of CC (Fig. 3B), may due to the saturation influence of binding sites on HSA for CC. For the convenience of calculation, we studied the quenching mechanism of fluorescence of HSA by LA (or CC) at low concentrations. The smaller plots (inserted in Fig. 3A and B) of F0/F with lower concentrations of LA (1.3 106e5.2 106 M) and CC (0.14 106e0.98 106 M), respectively, are linear and their corresponding results are listed in Table 1. The results show that Ksv is inversely correlated with temperature, and the values of kq (Table 1) are much greater than the maximum diffusion collision quenching rate constant (2.0 1010 M1 s1) of a variety of quenchers with biopolymer [20,21]. All the evidences confirm that a static quenching mechanism is responsible for the fluorescence quenching of HSA with LA (or CC) at low concentrations with
Fig. 3. SterneVolmer curves of HSA fluorescence quenched by LA (A) and CC (B).
complex formation [22]. Since the CC quenching constants Ksv are greater than that of LA in the same experimental conditions, quenching efficiency of CC is greater than that of LA. Furthermore, in many cases, a compound is used together with another compound. LA and CC are the important bioactive constituents of Dragon's blood, and they are both affinitive to HSA. When low dose CC (0.17 10-6 M) was added to HSA solution in
Table 1 The quenching constant Ksv and correlation coefficient. Compound
T(K)
Ksv ( 104 M1)
kq ( 1012 M1 s1)
Ra
Loureirin A
300 305 310 300 305 310
9.85 7.98 6.24 17.69 14.95 12.15
9.85 7.98 6.24 17.69 14.95 12.15
0.999 0.998 0.998 0.996 0.996 0.994
Cochinchinenin C
a
R is the linear correlated coefficient.
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conjunction with several different doses of LA (0e26.4 106 M), a comparison of SterneVolmer curves of HSA fluorescence quenched by LA in the absence and presence CC reveals obvious differences (Fig. 4). These two curves are gradually away from each other with the increase of LA concentration, indicating that there is a synergistic interaction between LA and CC [23]. 3.2. UVevis spectra To identify the further quenching mechanism of the compound interactions with HSA, UVevis absorption techniques are performed. UVeVis absorption spectroscopy is a simple but effective method in detecting the conformational changes of proteins and the complex formation [24,25]. Different HSA absorption spectra with various amounts of LA (or CC) are shown in Fig. 5. The influences of LA (or CC) were eliminated by using reference solution corresponding concentration of LA (or CC). From Fig. 5A, it can be observed that with the increase of LA concentration, the intensity of the HSA absorption peak at 280 nm decreases, while an increase is observed from Fig. 5B upon CC addition. These results show that compound interactions with HSA form a groundestate complex and the fluorescence quenching is mainly the static quenching process [26]. Meanwhile, the maximum absorption peak at 280 nm was concerned with the polarity of the microenvironment around tyrosine and tryptophan residues of HSA [27]. Therefore, the interaction of compound with HSA leads to the change in the conformation of HSA and hence changes the polarity of the microenvironment around tyrosine and tryptophan residues. 3.3. Binding constant and the number of binding sites For the static quenching process, it is assumed that small molecules bind independently to a set of equivalent sites on a macromolecule. The number of binding sites is obtained based on the Eq. (2):
lg½ðF0 FÞ=F ¼ lg K þ n lg ½Q
(2)
And for n z 1, Eq. (2) can be rewritten to the following Eq. (3) [28]:
F0 =ðF0 FÞ ¼ 1 þ K 1 ½Q 1
(3)
where K and n are the binding constants and the number of binding sites, respectively. Fig. 6 and Fig. 7 show the corresponding doubleelogarithm curve and the modified SterneVolmer plots. The values of K and n at three different temperatures are listed in Table 2. It is found that the binding site number n of LA and CC are
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approximately equal to 1, which means that there are just one single binding site on HSA for LA (or CC). And the binding constant for the LA-HSA (or CC-HSA) system decreases with the temperature increase. Additionally, the K of CC-HSA system are much larger than that of LA-HSA system, indicating that CC has a much higher affinity than LA in binding to HSA. These results coincide with the SterneVolmer quenching constant and suggest that the binding constant between LA (or CC) and HSA is moderate. 3.4. Thermodynamic parameters and nature of binding forces The acting forces between a compound and a biomolecule may include hydrogen bonds, van der Waals forces, hydrophobic and electrostatic interactions, etc. [29]. The thermodynamic parameters of binding reaction are the major evidence for confirming the intermolecular forces. Therefore, the thermodynamic parameters were calculated to characterize the forces involved in the binding process. Since there is no significant change in temperature, enthalpy change (△H) can be regarded as a constant, then its value and entropy change (△S) value can be calculated from the following Eq. (4). The value of free energy change (△G) can be obtained from Eq. (5):
ln K ¼ DH=RT þ DS=R
(4)
DG ¼ DH TDS
(5)
where K is the binding constant at corresponding temperature and R is the gas constant (8.3145 J mol1 K1). The temperatures used are 300, 305 and 310 K. The data are reported in Table 3. According to the sign of △H and △S, the model of interaction between the compound and HSA can be concluded [30]:(1) △H > 0 and △S > 0, hydrophobic forces; (2) △H < 0 and △S < 0, van der Waals interactions and hydrogen bonds; (3) △H < 0 and △S > 0, electrostatic interactions. Therefore, the negative △H and △S values suggest that LA (or CC) bind to HSA is mainly through hydrogen bonds and van der Waals forces. The negative value of △G indicates that the reaction is spontaneous [31]. 3.5. Fluorescence resonance energy transfer between compound and HSA The binding distance (r) between the donor and the acceptor €rster theory. According to can be determined based on the Fo €rster's theory [32], if the emitted fluorescence from a donor is Fo absorbed by an acceptor, the energy transfer may occur from the donor to the acceptor. The energy transfer efficiency E is calculated by using the following Eq. (6):
E ¼ 1 F=F0 ¼ R60
. R60 þ r6
(6)
where F and F0 are the fluorescence HSA intensities in the presence and absence of compound. R0 is the critical distance when the transfer efficiency is 50%, and its value can be calculated using the following equation:
R60 ¼ 8:8 1025 K2 $F$n4 $J
Fig. 4. SterneVolmer curves of HSA fluorescence quenched by LA in the absence and presence of CC.
(7)
where K2 is spatial orientation factor between the emission dipole of the donor and the absorption dipole of acceptor, and its value is taken as 2/3 for random orientations, the fluorescence quantum yield F of the donor in the absence of acceptor is 0.118, the average refractive index n of the medium is 1.336 [33], and J is the overlap
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Fig. 5. UVevis absorption spectra of HSA in the presence of different LA (A) and CC (B) concentration, aeg: 0, 3, 6, 9, 12, 15 and 18 106 M; CHSA ¼ 1.5 106 M; T ¼ 300 K.
Fig. 6. Double-log plots of LA (A) and CC (B) quenching effect on HSA fluorescence.
Fig. 7. The modified SterneVolmer plots of HSA on the different temperature with LA (A) and CC (B).
Table 2 The binding constant K and the number of binding sites n of the HSAecompound system. Compound
T(K)
K ( 104 M1)
n
Ra
Loureirin A
300 305 310 300 305 310
12.91 9.51 8.56 25.32 18.62 12.32
0.88 0.93 0.86 0.78 0.83 0.97
0.999 0.999 0.997 0.998 0.996 0.996
Cochinchinenin C
a
Table 3 Thermodynamic parameters of compoundeHSA interaction. Compound
T(K)
△H (kJ mol1)
△G (kJ mol1)
Loureirin A
300 305 310 300 305 310
31.98
29.28 29.23 29.19 31.09 30.67 30.26
Cochinchinenin C
55.94
△S (J mol1 K1) 9
82.86
R is the linear correlated coefficient for the K value.
integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor and can be calculated by the following equation:
J¼
X
.X Flεl l4 Dl Fl Dl
(8)
Fl is the fluorescence intensity of the donor in the absence of the acceptor at wavelength l and εl is the molar absorption coefficient of the acceptor at l.
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Fig. 8. Overlap of the fluorescence spectra of HSA (a) and the absorption spectra of LA (b) and CC (c). CHSA ¼ CLA ¼ CCC ¼ 1.5 106 M.
The overlap between the absorption spectrum of LA (or CC) and the fluorescence emission spectrum of HSA are shown in Fig. 8. Hence, from Eqs. (6)e(8), the value of parameters of LA were J ¼ 1.7848 1016cm3 Lmol1, R0 ¼ 1.84 nm, E ¼ 0.0987 and r ¼ 2.66 nm. The value of parameters of CC were J ¼ 4.404 1015cm3 Lmol1, R0 ¼ 2.14 nm, E ¼ 0.1212 and r ¼ 2.975 nm. The donor-acceptor distances are less than 7 nm and 0.5R0 < r < 1.5R0, which indicates that the energy transfer occurs with high probability [34].
3.6. Effect of compound on the HSA conformation The synchronous fluorescence is a sensitive technique to study the effect of compound on the protein microenvironment and the
environment of amino acid residues. The possible shift at the maximum excitation wavelength of the amino acid residues corresponds to the changes of the polarity around chromophore molecule [35,36]. Therefore, the shift of the maximum excitation wavelength reveals the conformational change of HSA. Synchronous fluorescence spectra of HSA are obtained after simultaneously scanning excitation and emission monochromators. These spectra provide the characteristic information about the tyrosine or tryptophan residues when the Dl is set at 15 or 60 nm, respectively [37,38]. The synchronous fluorescence spectra of HSA upon addition of compound at Dl ¼ 15 and 60 nm are displayed in Fig. 9. When Dl is set to 15 nm, it is apparent from Fig. 9A that the maximum excitation wavelength had a slight red shift (288.5 / 291 nm) in LA-HSA system; While in CC-HSA system,
Fig. 9. Synchronous fluorescence spectra of HSAecompound system. (A) (C) Dl ¼ 15 nm; (B) (D) Dl ¼ 60 nm, CHSA ¼ 1.5 106 M; CLA ¼ CCC, aef: 0, 0.14, 0.28, 0.42, 0.56 and 0.7 106 M (T ¼ 310 K).
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Fig. 10. Three-dimensional fluorescence spectra (AeC) and corresponding contour maps (A0 eC0 ) of HSA (A and A0 ), LA-HSA systems (B and B0 ) and CC-HSA systems (C and C0 ); CHSA ¼ 1.5 106 M.
there is almost no fluorescence quenching in Fig. 9C. When Dl ¼ 60 nm, the maximum excitation wavelength yields a moderate red shift (286.5 / 290.5 nm) in LA-HSA system (Fig. 9B); in CC-HSA system, a slight blue shift (288 / 287 nm) is observed from Fig. 9D. Obviously, the quenching of tryptophan residue is stronger than that of tyrosine residue, suggesting that tryptophan residues contribute greatly to the quenching of intrinsic fluorescence of HSA [31]. Moreover, the red-shift effects suggested that the interaction between LA and HSA increases the polarity around the tyrosine and tryptophan residues and decreases the hydrophobicity. While the binding of CC to HSA had little effect on the microenvironment around the alone tryptophan residue (Trp-214), from polar to slightly nonpolar [39]. These results suggested that the conformations of the HSA have been changed on the addition of LA or CC. In order to investigate the conformation change of HSA by compounds more carefully, three-dimensional fluorescence spectra are applied to observe the changes in the secondary structure of HSA. The 3-D fluorescence spectra and the corresponding contour maps of HSA (A and A0 ) and HSA in the presence of LA (B and B0 ) and CC (C and C0 ) with compound/HSA molar ratios of 2:1 are displayed
in Fig. 10. Peak 1 (lex ¼ 280 nm, lem ¼ 348 nm) represented the intrinsic fluorescence characteristics of HSA due to tryptophan and tyrosine residues and reflected changes in the tertiary structure of HSA upon complexation with compound [40]. Addition of LA to HSA produced an obvious decrease in the intensity along with a slight red shift in peaks 1, being ~28%, 2.5 nm. And an obvious decrease in the fluorescence intensity of HSA and a slight blue shift can be seen in peaks 1 after addition of CC, being ~14%, 1 nm. The changes in the 3-D fluorescence spectra characteristics are similar to synchronous fluorescence results (Fig. 9), which demonstrates that some microenvironmental and conformational changes in HSA have been slightly altered in the presence of LA (or CC) (see Fig. 10). 3.7. Molecular docking study The Discovery Studio program was used to study the interaction between compounds (LA，CC) and HSA, the most possible binding mode is shown in Fig. 11. As seen in Fig. 11A, LA can bind to a hydrophobic pocket of subdomain IIA of HSA, the binding site were obtained as Lys195, Lys199, Trp214, Arg218, Leu219, Arg222, Phe223, Leu238, His242, Leu260, Ala261, Ile264, Ser287, Ile290,
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Fig. 11. (A) (C) Compound docked in the binding pocket of HSA. The ligand structure is represented using stick model, and the residues of the HSA are presented using line; (B) (D) Two-dimensional schematic representation of hydrogen bond interaction. Hydrogen bond depicted in dashed line.
Ala291, Glu292 and Asp451. From Fig. 11C, CC is located at the binding pocket between subdomain IIA and IIB of HSA, also the binding site were obtained as Glu208, Arg209, Lys212, Ala213, Trp214, Val216, Val231,Ser232, Val235, Thr236, Ala322, Lys323, Asp324, Val325, Leu327, Gly328, Leu331, Ala350, Lys351, Glu354. Furthermore, it is seen that LA and CC are adjacent to Trp214, which explain the efficient fluorescence quenching of HSA emission in the presence of LA and CC (as shown in Fig. 2). Three hydrogen bonds interactions between HSA and LA were observed in Fig. 11B, while only one hydrogen bonding interaction was observed between the hydroxyl (OH) group of CC and Lys323 of HSA (Fig.D). And there are also a number of van der Waals forces, which play an important role in stabilizing the molecule. The results indicated that the interaction between LA (or CC) and HSA was dominated by van der Waals forces as well as hydrogen bonds, which was consistent with our experimental results.
4. Conclusions In this paper, fluorescence and UVevis absorption spectroscopic methods were applied to study the interaction of HSA with LA (or CC) at different temperatures under physiological conditions. The results showed that LA and CC had a strong ability to quench the HSA intrinsic fluorescence through a combined quenching (dynamic and static quenching), while the mechanism reveals a static quenching at low concentrations. Moreover, there is a synergistic interaction between LA and CC and the quenching efficiency, binding affinity of CC to HSA were more than that of LA at low concentrations. Meanwhile the thermodynamic parameters indicated that the binding reactions of HSA with LA and CC were spontaneous and caused by hydrogen bonding interactions and van
der Waals force. The value of binding sites (n) for LA and CC were found to be about 1. From the fluorescence energy transfer studies, the transfer distance from tryptophan-214 of HSA to LA and CC were calculated as 2.66 nm and 2.975 nm respectively. Furthermore, the results of synchronous fluorescence and threedimensional fluorescence spectroscopy showed that the binding of LA (or CC) to HSA would lead to a slight conformational change in HSA. The molecular modeling study results in binding forces were much coincided with the experimental ones. This study is expected to provide important insight into the interactions of HSA with drugs and the results have a great significance in efficacy comparison of these bioactive constituents, which will be helpful to drug design and drug screening research. Acknowledgments The authors wish to thank Pro. Pejmen HADI and Dr. Yuan-shuai Liu and Zhu-yi Jin from Hong Kong University of Science and Technology for their kindly technical guidance. We also thank Dr. zhirui Liu from Shanghai Jiaotong University School of Medicine for supporting the molecular modeling software (Discovery Studio 4.0). This study was supported by the National Key Technology Support Program of China (No. 2013BAD12B06) and the experimental apparatus were provided by experimental center for life sciences of Shanghai University. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.ejmech.2015.02. 025. These data include MOL files and InChiKeys of the most
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1089e1101. [19] F. Mohammadi, A.K. Bordbar, A. Divsalar, et al., Protein J. 28 (2009) 117e123. [20] Q.J. Zhou, J.F. Xiang, Y.L. Tang, et al., Colloids Surf. B Biointerfaces 61 (2008) 75e80. [21] A. Gerbanowski, C. Malabat, C. Rabiller, et al., Food Chem. 47 (1999) 5218e5226. [22] G.K. Wang, D.C. Wang, X. Li, et al., Colloids Surf. B Biointerfaces 84 (2011) 272e279. [23] C. Miaskowski, K.A. Sutters, Y.O. Taiwo, et al., Pain 49 (1992) 137e144. [24] S.Y. Bi, D.Q. Song, Y. Tian, et al., Spectrochim. Acta Part A 61 (2005) 629e636. [25] P.B. Kandagal, S. Ashoka, J. Seetharamappa, et al., J. Pharm. Biomed. Anal. 41 (2006) 393e399. [26] B.S. Liu, C.L. Xue, J. Wang, et al., J. Lumin 130 (2010) 1999e2003. [27] D.J. Li, M. Zhu, C. Xu, et al., Eur. J. Med. Chem. 46 (2011) 588e599. [28] M.X. Xie, X.Y. Xu, Y.D. Wang, Biochim. Biophys. Acta 1724 (2005) 215e224. [29] I.M. Klotz, Ann. N. Y. Acad. Sci. 226 (1993) 18e35. [30] Y.J. Hu, H.G. Yu, J.X. Dong, et al., Spectrochim. Acta. A 65 (2006) 988. [31] F. Samari, M. Shamsipur, B. Hemmateenejad, et al., Eur. J. Med. Chem. 54 (2012) 255e263. €rster, O. Sinanoglu (Eds.), Modern Quantum Chemistry, vol. 3, Academic [32] T. Fo Press, New York, 1996, pp. 93e138. [33] C.B. Berde, B.S. Hudson, R.D. Simoni, et al., J. Biol. Chem. 254 (1979) 391e400. [34] W.Y. He, Y. Li, C.X. Xue, et al., Bioorgan. Med. Chem. 13 (2005) 1837e1845. [35] T. Yuan, A.M. Weljie, H.J. Vogel, Biochemistry 37 (1998) 3187e3195. [36] Y.Q. Wang, H.M. Zhang, G.C. Zhang, et al., J. Mol. Struct. 886 (2008) 77e84. [37] W.C. Abert, W.M. Gregory, G.S. Allan, Anal. Biochem. 213 (1993) 407e413. [38] G.W. Zhang, Q.M. Que, J.H. Pan, et al., J. Mol. Struct. 881 (2008) 132e138. [39] K.H. Ulrich, Pharmacol. Rev. 33 (1981) 17e53. [40] S.R. Feroz, S.B. Mohamad, N. Bujang, et al., J. Agric. Food Chem. 60 (2012) 5899e5908.
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Comparison between loureirin A and cochinchinenin C on the interaction with human serum albumin Xu Chen a, 1, Kai Qian b, 1, Qin Chen b, * a b
Experimental Center for Life Scinence, Shanghai University, Shanghai, PR China Shanghai Key Lab Bioenergy Crops, School of Life Sciences, Shanghai University, Shanghai, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 April 2014 Received in revised form 16 February 2015 Accepted 17 February 2015 Available online 24 February 2015
The interactions of loureirin A (LA) and cochinchinenin C (CC) with human serum albumin (HSA) under simulated physiological conditions (pH ¼ 7.4) have been studied with fluorescence, UVevis absorption spectroscopic method and molecular docking technique. The results indicated that there was a synergistic interaction between LA and CC, and the fluorescence quenching of HSA by LA (or CC) was a combined quenching procedure (dynamic and static quenching). At low compound concentrations, the quenching constants KSV of CC was larger than that of LA, which meant the CC efficacy may be better than that of LA. The negative △H and △S values suggested hydrogen bonds and van der Waals forces played the major role in the binding of LA (or CC) to HSA. The efficiency of energy transfer and distance between the compounds and HSA was calculated. Moreover, the results of synchronous and three-dimensional fluorescence demonstrated that the HSA microenvironment was changed in the presence of LA (or CC). Finally, the binding of LA (or CC) to HSA was modeled by molecular docking, which is in good accordance with the experimental studies. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Loureirin A Cochinchinenin C Human serum albumin Fluorescence quenching Molecular docking
1. Introduction Loureirin A (LA) and cochinchinenin C (CC) (structures shown in Fig. 1), which belong to flavonoids, are the important bioactive constituents of Dragon's blood [1]. As is well-known, Dragon's blood is one of the renowned traditional medicines, of which from Dranaena cochinchinensis (Lour). S.C. Chen is named ‘Resina Draaconis’ in China. Dragon's blood has been widely used for diverse biological applications including antithrombotic, anti-blood stasis, anticoagulant, analgesic, blood circulation promotion, reducing high level of blood sugar and other important therapeutic activities [2e7]. And there are different affinity and activity between LA and CC, which can be attributed to their different molecular structures and different molecular weights. In previous studies, LA was reported to exhibit analgesic activity [1], and CC was reported with anti-Helicobacter pylori and thrombin inhibitory activities [8]. Human serum albumin (HSA) is the most abundant protein in plasma and has many physiological functions. The principle function of HSA is to act as a carrier for many endogenous and
* Corresponding author. E-mail address: [email protected] (Q. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2015.02.025 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
exogenous molecules and metabolites to particular biological targets [9]. Therefore, HSA plays an important role in the distribution, metabolism, efficacy and interaction with the drug target tissues [10]. The drug pharmaceutical activity level depends on the interaction with targeting molecules, mode of action and affinity size, while the structural differences in drug often lead to changes in its biological activity [11,12]. Thus, the investigation of drugs binding to HSA is of important significance [13]. In the present work, a comparative study of the interaction between HSA and LA (or CC) was undertaken by fluorescence and UVevis absorption spectroscopic and docking simulation methods. The spectroscopic techniques, which are sensitive and relatively simple, are often used to monitor the molecular interactions [14,15]. The results obtained from these methods not only revealed the LA (or CC) and HSA interaction mechanism, but also may provide some valuable information for drug design and pharmaceutical research with comparative studies on LA and CC interactions with HSA. 2. Experimental method 2.1. Materials Human serum albumin (approximately 99% and fatty acid free)
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Fig. 1. Molecular structures of loureirin A (A) and cochinchinenin C (B).
was purchased from Sigma Chemical Company (USA), loureirin A and cochinchinenin C were purchased from National Institute for the Control of Pharmaceutical and Biological Products (China). The stock solution of human serum albumin was prepared in 0.1 M TriseHCl buffer with pH ¼ 7.4 containing 0.1 M sodium chloride. A 1.5 106 M working solution of HSA was prepared by appropriate dilution of the stock solution. Loureirin A was directly dissolved in anhydrous alcohol as stock solution (2.0 103 M). Stock solution of cochinchinenin C (0.4 103 M) was also prepared by dissolving appropriate amount of the drugs in anhydrous alcohol. All other reagents were of guarantee grade and double distilled water was used throughout. 2.2. Apparatus All fluorescence measurements were performed on a Spectrofluorimeter Model LS-55 (PerkinElmer, USA) equipped with a quartz cell (10-mm) and a thermostat bath. The absorption spectra were recorded on Cary100 UVevis spectrophotometer (Varian, Australia) with a 10-mm quartz cuvette at room temperature. The pH values were measured on a pHS-3C Digital pH-meter (Shanghai Lei Ci device Works, China).
were obtained upon excitation wavelength range of 200e480 nm with 10 nm increments and monitoring the emission spectra from 220 to 500 nm. 2.3.2. UVevis absorption spectra The absorption titration were carried out by keeping the concentration of HSA in 1.5 106 M, while the LA (or CC) concentration was varied from 0 to 18 106 M. Changes in absorbance at 280 nm are convincing evidences for the formation of compoundeHSA complex. The absorption spectra of LA and CC (1.5 106 M) were also recorded. 2.3.3. Molecular docking The crystal structure of the HSA (PDB ID: 1h9z) was obtained from the protein data bank. Water molecules were removed, and hydrogen atoms were added. The structure of LA and CC were created as mol files using ChemDraw Ultra (Ver. 9.0). All simulations were performed by using Discovery Studio 4.0 software, which is an interactive molecular graphics program assisting the understanding of the compoundeprotein interaction. 3. Results and discussion
2.3. Procedures
3.1. Fluorescence quenching of HSA
2.3.1. Fluorescence spectra 3.0 mL solution, containing 1.5 106 M HSA, was titrated by successive additions of 1.0 106 L LA (or CC) stock solution. The solution was allowed to stand for 5 min to equilibrate. Fluorescence emission spectra were recorded from 300 to 450 nm with an excitation wavelength at 280 nm at three different temperatures (300, 305 and 310 K). On the other hand, the D-value between excitation and emission wavelengths (Dl) was set at 15 and 60 nm for the synchronous fluorescence spectra measurements, respectively. Excitation wavelength was set from 270 to 320 nm. Both excitation and emission bandwidths were set at 10 nm. Three-dimensional (3-D) fluorescence spectra of HSA (1.5 106 M) and the compoundHSA complexes (2:1 M ratios)
The fluorescence emission spectra of HSA in the presence of different LA and CC concentrations are recorded at three temperatures (300, 305 and 310 K) and one of them (310 K) are shown in Fig. 2. The excitation wavelength was set at 280 nm, so that both tyrosyl and tryptophyl fluorophores were excited [16]. Fig. 2 suggests that HSA exhibits a stronger fluorescence peak, while LA and CC display weaker intrinsic fluorescence under the same experimental conditions. Furthermore, the fluorescence intensity of HSA drops gradually with the increase of LA concentration, along with a slight red shift (347.5 / 348.5 nm). With the increase of CC concentration, binding sites on HSA are increasingly saturated, which can be explained by the presence of aggregates of CC. The resulting steric hindrance may take place that weaken the affinity of CC to
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Fig. 2. Emission spectra of HSA with LA (A) and CC (B). CHSA ¼ 1.5 106 M, C LA, a / h: 0, 0.65, 1.3, 1.95, 2.6, 3.25, 3.9 and 4.55 106 M; C CC, a / h: 0, 0.14, 0.28, 0.42, 0.56, 0.7, 0.84, and 0.98 106 M; T ¼ 310 K.
HSA. As a result, the fluorescence intensity of HSA is almost no longer reduced and the position of the emission maximum wavelength shows a moderate blue shift (346.5 / 344 nm). Therefore, the observation indicates that the intrinsic fluorescence of HSA is quenched accompanying with a microenvironmental change around HSA after the addition of LA (or CC). Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interactions with a quencher molecule, such as excited-state reactions, molecular rearrangement, energy transfer, groundestate complex formation, and collisional quenching. Quenching can be induced by dynamic and static processes, which are caused by collisional encountering between the fluorophore and quencher and non-fluorescent ground state complex formation, respectively. However, in many instances, the fluorophore can be quenched both by collisions and by complex formation with the same quencher. The characteristic feature of the SterneVolmer plot exhibits an upward curvature for the distinct situation, concave towards the y axis at high quencher concentration [17]. To confirm the quenching mechanism, the fluorescence quenching of HSA at different temperatures (300, 305 and 310 K) has been analyzed by SterneVolmer equation:
F0 =F ¼ 1 þ kq t0 ½Q ¼ 1 þ Ksv Q
(1)
where F0 and F represent the fluorescence intensities of HSA in absence and presence of quencher, respectively, [Q] is the quencher concentration, kq is bimolecular quenching rate constant, Ksv is the SterneVolmer quenching constant and t0 is the average lifetime of the biomolecule without quencher (t0 ¼ 108 s) [18,19]. Fig. 3 displays the SterneVolmer plots for the fluorescence quenching of HSA by LA and CC at different temperatures. In Fig. 3A, the upward curves at high concentrations suggest that the fluorescence quenching mechanism of LA with HSA is a combined one. The plateau at high concentrations of CC (Fig. 3B), may due to the saturation influence of binding sites on HSA for CC. For the convenience of calculation, we studied the quenching mechanism of fluorescence of HSA by LA (or CC) at low concentrations. The smaller plots (inserted in Fig. 3A and B) of F0/F with lower concentrations of LA (1.3 106e5.2 106 M) and CC (0.14 106e0.98 106 M), respectively, are linear and their corresponding results are listed in Table 1. The results show that Ksv is inversely correlated with temperature, and the values of kq (Table 1) are much greater than the maximum diffusion collision quenching rate constant (2.0 1010 M1 s1) of a variety of quenchers with biopolymer [20,21]. All the evidences confirm that a static quenching mechanism is responsible for the fluorescence quenching of HSA with LA (or CC) at low concentrations with
Fig. 3. SterneVolmer curves of HSA fluorescence quenched by LA (A) and CC (B).
complex formation [22]. Since the CC quenching constants Ksv are greater than that of LA in the same experimental conditions, quenching efficiency of CC is greater than that of LA. Furthermore, in many cases, a compound is used together with another compound. LA and CC are the important bioactive constituents of Dragon's blood, and they are both affinitive to HSA. When low dose CC (0.17 10-6 M) was added to HSA solution in
Table 1 The quenching constant Ksv and correlation coefficient. Compound
T(K)
Ksv ( 104 M1)
kq ( 1012 M1 s1)
Ra
Loureirin A
300 305 310 300 305 310
9.85 7.98 6.24 17.69 14.95 12.15
9.85 7.98 6.24 17.69 14.95 12.15
0.999 0.998 0.998 0.996 0.996 0.994
Cochinchinenin C
a
R is the linear correlated coefficient.
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conjunction with several different doses of LA (0e26.4 106 M), a comparison of SterneVolmer curves of HSA fluorescence quenched by LA in the absence and presence CC reveals obvious differences (Fig. 4). These two curves are gradually away from each other with the increase of LA concentration, indicating that there is a synergistic interaction between LA and CC [23]. 3.2. UVevis spectra To identify the further quenching mechanism of the compound interactions with HSA, UVevis absorption techniques are performed. UVeVis absorption spectroscopy is a simple but effective method in detecting the conformational changes of proteins and the complex formation [24,25]. Different HSA absorption spectra with various amounts of LA (or CC) are shown in Fig. 5. The influences of LA (or CC) were eliminated by using reference solution corresponding concentration of LA (or CC). From Fig. 5A, it can be observed that with the increase of LA concentration, the intensity of the HSA absorption peak at 280 nm decreases, while an increase is observed from Fig. 5B upon CC addition. These results show that compound interactions with HSA form a groundestate complex and the fluorescence quenching is mainly the static quenching process [26]. Meanwhile, the maximum absorption peak at 280 nm was concerned with the polarity of the microenvironment around tyrosine and tryptophan residues of HSA [27]. Therefore, the interaction of compound with HSA leads to the change in the conformation of HSA and hence changes the polarity of the microenvironment around tyrosine and tryptophan residues. 3.3. Binding constant and the number of binding sites For the static quenching process, it is assumed that small molecules bind independently to a set of equivalent sites on a macromolecule. The number of binding sites is obtained based on the Eq. (2):
lg½ðF0 FÞ=F ¼ lg K þ n lg ½Q
(2)
And for n z 1, Eq. (2) can be rewritten to the following Eq. (3) [28]:
F0 =ðF0 FÞ ¼ 1 þ K 1 ½Q 1
(3)
where K and n are the binding constants and the number of binding sites, respectively. Fig. 6 and Fig. 7 show the corresponding doubleelogarithm curve and the modified SterneVolmer plots. The values of K and n at three different temperatures are listed in Table 2. It is found that the binding site number n of LA and CC are
495
approximately equal to 1, which means that there are just one single binding site on HSA for LA (or CC). And the binding constant for the LA-HSA (or CC-HSA) system decreases with the temperature increase. Additionally, the K of CC-HSA system are much larger than that of LA-HSA system, indicating that CC has a much higher affinity than LA in binding to HSA. These results coincide with the SterneVolmer quenching constant and suggest that the binding constant between LA (or CC) and HSA is moderate. 3.4. Thermodynamic parameters and nature of binding forces The acting forces between a compound and a biomolecule may include hydrogen bonds, van der Waals forces, hydrophobic and electrostatic interactions, etc. [29]. The thermodynamic parameters of binding reaction are the major evidence for confirming the intermolecular forces. Therefore, the thermodynamic parameters were calculated to characterize the forces involved in the binding process. Since there is no significant change in temperature, enthalpy change (△H) can be regarded as a constant, then its value and entropy change (△S) value can be calculated from the following Eq. (4). The value of free energy change (△G) can be obtained from Eq. (5):
ln K ¼ DH=RT þ DS=R
(4)
DG ¼ DH TDS
(5)
where K is the binding constant at corresponding temperature and R is the gas constant (8.3145 J mol1 K1). The temperatures used are 300, 305 and 310 K. The data are reported in Table 3. According to the sign of △H and △S, the model of interaction between the compound and HSA can be concluded [30]:(1) △H > 0 and △S > 0, hydrophobic forces; (2) △H < 0 and △S < 0, van der Waals interactions and hydrogen bonds; (3) △H < 0 and △S > 0, electrostatic interactions. Therefore, the negative △H and △S values suggest that LA (or CC) bind to HSA is mainly through hydrogen bonds and van der Waals forces. The negative value of △G indicates that the reaction is spontaneous [31]. 3.5. Fluorescence resonance energy transfer between compound and HSA The binding distance (r) between the donor and the acceptor €rster theory. According to can be determined based on the Fo €rster's theory [32], if the emitted fluorescence from a donor is Fo absorbed by an acceptor, the energy transfer may occur from the donor to the acceptor. The energy transfer efficiency E is calculated by using the following Eq. (6):
E ¼ 1 F=F0 ¼ R60
. R60 þ r6
(6)
where F and F0 are the fluorescence HSA intensities in the presence and absence of compound. R0 is the critical distance when the transfer efficiency is 50%, and its value can be calculated using the following equation:
R60 ¼ 8:8 1025 K2 $F$n4 $J
Fig. 4. SterneVolmer curves of HSA fluorescence quenched by LA in the absence and presence of CC.
(7)
where K2 is spatial orientation factor between the emission dipole of the donor and the absorption dipole of acceptor, and its value is taken as 2/3 for random orientations, the fluorescence quantum yield F of the donor in the absence of acceptor is 0.118, the average refractive index n of the medium is 1.336 [33], and J is the overlap
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Fig. 5. UVevis absorption spectra of HSA in the presence of different LA (A) and CC (B) concentration, aeg: 0, 3, 6, 9, 12, 15 and 18 106 M; CHSA ¼ 1.5 106 M; T ¼ 300 K.
Fig. 6. Double-log plots of LA (A) and CC (B) quenching effect on HSA fluorescence.
Fig. 7. The modified SterneVolmer plots of HSA on the different temperature with LA (A) and CC (B).
Table 2 The binding constant K and the number of binding sites n of the HSAecompound system. Compound
T(K)
K ( 104 M1)
n
Ra
Loureirin A
300 305 310 300 305 310
12.91 9.51 8.56 25.32 18.62 12.32
0.88 0.93 0.86 0.78 0.83 0.97
0.999 0.999 0.997 0.998 0.996 0.996
Cochinchinenin C
a
Table 3 Thermodynamic parameters of compoundeHSA interaction. Compound
T(K)
△H (kJ mol1)
△G (kJ mol1)
Loureirin A
300 305 310 300 305 310
31.98
29.28 29.23 29.19 31.09 30.67 30.26
Cochinchinenin C
55.94
△S (J mol1 K1) 9
82.86
R is the linear correlated coefficient for the K value.
integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor and can be calculated by the following equation:
J¼
X
.X Flεl l4 Dl Fl Dl
(8)
Fl is the fluorescence intensity of the donor in the absence of the acceptor at wavelength l and εl is the molar absorption coefficient of the acceptor at l.
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Fig. 8. Overlap of the fluorescence spectra of HSA (a) and the absorption spectra of LA (b) and CC (c). CHSA ¼ CLA ¼ CCC ¼ 1.5 106 M.
The overlap between the absorption spectrum of LA (or CC) and the fluorescence emission spectrum of HSA are shown in Fig. 8. Hence, from Eqs. (6)e(8), the value of parameters of LA were J ¼ 1.7848 1016cm3 Lmol1, R0 ¼ 1.84 nm, E ¼ 0.0987 and r ¼ 2.66 nm. The value of parameters of CC were J ¼ 4.404 1015cm3 Lmol1, R0 ¼ 2.14 nm, E ¼ 0.1212 and r ¼ 2.975 nm. The donor-acceptor distances are less than 7 nm and 0.5R0 < r < 1.5R0, which indicates that the energy transfer occurs with high probability [34].
3.6. Effect of compound on the HSA conformation The synchronous fluorescence is a sensitive technique to study the effect of compound on the protein microenvironment and the
environment of amino acid residues. The possible shift at the maximum excitation wavelength of the amino acid residues corresponds to the changes of the polarity around chromophore molecule [35,36]. Therefore, the shift of the maximum excitation wavelength reveals the conformational change of HSA. Synchronous fluorescence spectra of HSA are obtained after simultaneously scanning excitation and emission monochromators. These spectra provide the characteristic information about the tyrosine or tryptophan residues when the Dl is set at 15 or 60 nm, respectively [37,38]. The synchronous fluorescence spectra of HSA upon addition of compound at Dl ¼ 15 and 60 nm are displayed in Fig. 9. When Dl is set to 15 nm, it is apparent from Fig. 9A that the maximum excitation wavelength had a slight red shift (288.5 / 291 nm) in LA-HSA system; While in CC-HSA system,
Fig. 9. Synchronous fluorescence spectra of HSAecompound system. (A) (C) Dl ¼ 15 nm; (B) (D) Dl ¼ 60 nm, CHSA ¼ 1.5 106 M; CLA ¼ CCC, aef: 0, 0.14, 0.28, 0.42, 0.56 and 0.7 106 M (T ¼ 310 K).
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Fig. 10. Three-dimensional fluorescence spectra (AeC) and corresponding contour maps (A0 eC0 ) of HSA (A and A0 ), LA-HSA systems (B and B0 ) and CC-HSA systems (C and C0 ); CHSA ¼ 1.5 106 M.
there is almost no fluorescence quenching in Fig. 9C. When Dl ¼ 60 nm, the maximum excitation wavelength yields a moderate red shift (286.5 / 290.5 nm) in LA-HSA system (Fig. 9B); in CC-HSA system, a slight blue shift (288 / 287 nm) is observed from Fig. 9D. Obviously, the quenching of tryptophan residue is stronger than that of tyrosine residue, suggesting that tryptophan residues contribute greatly to the quenching of intrinsic fluorescence of HSA [31]. Moreover, the red-shift effects suggested that the interaction between LA and HSA increases the polarity around the tyrosine and tryptophan residues and decreases the hydrophobicity. While the binding of CC to HSA had little effect on the microenvironment around the alone tryptophan residue (Trp-214), from polar to slightly nonpolar [39]. These results suggested that the conformations of the HSA have been changed on the addition of LA or CC. In order to investigate the conformation change of HSA by compounds more carefully, three-dimensional fluorescence spectra are applied to observe the changes in the secondary structure of HSA. The 3-D fluorescence spectra and the corresponding contour maps of HSA (A and A0 ) and HSA in the presence of LA (B and B0 ) and CC (C and C0 ) with compound/HSA molar ratios of 2:1 are displayed
in Fig. 10. Peak 1 (lex ¼ 280 nm, lem ¼ 348 nm) represented the intrinsic fluorescence characteristics of HSA due to tryptophan and tyrosine residues and reflected changes in the tertiary structure of HSA upon complexation with compound [40]. Addition of LA to HSA produced an obvious decrease in the intensity along with a slight red shift in peaks 1, being ~28%, 2.5 nm. And an obvious decrease in the fluorescence intensity of HSA and a slight blue shift can be seen in peaks 1 after addition of CC, being ~14%, 1 nm. The changes in the 3-D fluorescence spectra characteristics are similar to synchronous fluorescence results (Fig. 9), which demonstrates that some microenvironmental and conformational changes in HSA have been slightly altered in the presence of LA (or CC) (see Fig. 10). 3.7. Molecular docking study The Discovery Studio program was used to study the interaction between compounds (LA，CC) and HSA, the most possible binding mode is shown in Fig. 11. As seen in Fig. 11A, LA can bind to a hydrophobic pocket of subdomain IIA of HSA, the binding site were obtained as Lys195, Lys199, Trp214, Arg218, Leu219, Arg222, Phe223, Leu238, His242, Leu260, Ala261, Ile264, Ser287, Ile290,
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Fig. 11. (A) (C) Compound docked in the binding pocket of HSA. The ligand structure is represented using stick model, and the residues of the HSA are presented using line; (B) (D) Two-dimensional schematic representation of hydrogen bond interaction. Hydrogen bond depicted in dashed line.
Ala291, Glu292 and Asp451. From Fig. 11C, CC is located at the binding pocket between subdomain IIA and IIB of HSA, also the binding site were obtained as Glu208, Arg209, Lys212, Ala213, Trp214, Val216, Val231,Ser232, Val235, Thr236, Ala322, Lys323, Asp324, Val325, Leu327, Gly328, Leu331, Ala350, Lys351, Glu354. Furthermore, it is seen that LA and CC are adjacent to Trp214, which explain the efficient fluorescence quenching of HSA emission in the presence of LA and CC (as shown in Fig. 2). Three hydrogen bonds interactions between HSA and LA were observed in Fig. 11B, while only one hydrogen bonding interaction was observed between the hydroxyl (OH) group of CC and Lys323 of HSA (Fig.D). And there are also a number of van der Waals forces, which play an important role in stabilizing the molecule. The results indicated that the interaction between LA (or CC) and HSA was dominated by van der Waals forces as well as hydrogen bonds, which was consistent with our experimental results.
4. Conclusions In this paper, fluorescence and UVevis absorption spectroscopic methods were applied to study the interaction of HSA with LA (or CC) at different temperatures under physiological conditions. The results showed that LA and CC had a strong ability to quench the HSA intrinsic fluorescence through a combined quenching (dynamic and static quenching), while the mechanism reveals a static quenching at low concentrations. Moreover, there is a synergistic interaction between LA and CC and the quenching efficiency, binding affinity of CC to HSA were more than that of LA at low concentrations. Meanwhile the thermodynamic parameters indicated that the binding reactions of HSA with LA and CC were spontaneous and caused by hydrogen bonding interactions and van
der Waals force. The value of binding sites (n) for LA and CC were found to be about 1. From the fluorescence energy transfer studies, the transfer distance from tryptophan-214 of HSA to LA and CC were calculated as 2.66 nm and 2.975 nm respectively. Furthermore, the results of synchronous fluorescence and threedimensional fluorescence spectroscopy showed that the binding of LA (or CC) to HSA would lead to a slight conformational change in HSA. The molecular modeling study results in binding forces were much coincided with the experimental ones. This study is expected to provide important insight into the interactions of HSA with drugs and the results have a great significance in efficacy comparison of these bioactive constituents, which will be helpful to drug design and drug screening research. Acknowledgments The authors wish to thank Pro. Pejmen HADI and Dr. Yuan-shuai Liu and Zhu-yi Jin from Hong Kong University of Science and Technology for their kindly technical guidance. We also thank Dr. zhirui Liu from Shanghai Jiaotong University School of Medicine for supporting the molecular modeling software (Discovery Studio 4.0). This study was supported by the National Key Technology Support Program of China (No. 2013BAD12B06) and the experimental apparatus were provided by experimental center for life sciences of Shanghai University. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.ejmech.2015.02. 025. These data include MOL files and InChiKeys of the most
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