Research article Received: 18 February 2014,
Revised: 16 May 2014,
Accepted: 22 May 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2724
Spectroscopy and docking simulations of the interaction between lochnericine and bovine serum albumin Qing Wang, Jiawei He, Jin Yan, Di Wu and Hui Li* ABSTRACT: Lochnericine (LOC) is a component of Voacanga africana, which is a type of traditional medical food in Africa widely used for treating diseases. In this article, the interaction between LOC and bovine serum albumin (BSA) was studied by fluorescence spectroscopy. Furthermore, Fourier transform infrared (FTIR), Raman and circular dichroism (CD) were used to investigate the structural changes of BSA. The experimental results consistently indicated that LOC changed the secondary structure of BSA. Three structure-similar components were used to study the interference experiments. The molecular modeling results showed that LOC could bind within not only sites I and II, but also bind the cavity of subdomain IB. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: lochnericine; bovine serum albumin (BSA); interaction; structural changes; molecular modeling
Introduction Alkaloids are a group of naturally occurring chemical compounds that mostly contain basic nitrogen atoms, and have diverse and important physiological effects on humans and other animals (1). Therefore, alkaloids have been the subject of much clinical research, and traditional medical foods rich in alkaloids have attracted interest in recent years (2,3). Alkaloids are the main active components of Voacanga africana (4). In Africa, V. africana is used to treat diseases like leprosy, diarrhea, generalized edema, convulsions in children, orchitis and ectopic testes gonorrhea (5). Lochnericine (LOC), which is a type of indole alkaloid, can be extracted from V. africana. Although LOC was discovered long ago and its structure (Fig. 1) has been identified previously (6,7), it has not been used as a single drug. Interactions between potential medical LOC and serum albumin must be considered because of the relevance to its transport, biological activity and clearance. Serum albumin, the most abundant protein present in the circulatory system of animals, is a major macromolecule that contributes to osmotic blood pressure. Serum albumin can bind various compounds (8,9). When drugs are absorbed into the circulatory system, they can extensively and reversibly bind to serum albumin. The interaction between drug and protein can significantly affect the biological activity and toxicity of drugs in pharmacology (10–12). Usually, the activity of a protein is closely related to its conformation and structure, and gives information critical to understanding the relationship between structure and function (13,14). Thus, the identification of a protein through its conformational features is essential in the medical applications (15). Fourier transform infrared (FTIR) spectroscopy, Raman scattering and circular dichroism (CD) spectroscopy are powerful tools for investigating structural changes in serum albumin (16–19). They provide important information on both the secondary and tertiary structures of proteins, as well as on the protein microenvironment (20). Bovine serum albumin (BSA) was selected as the model protein in this work due to its abundance, low cost, ease of purification and
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stability. The results of all the BSA studies were consistent with the fact that human and bovine serum albumins are homologous proteins (21,22). In this work, air-dried seeds of V. africana stapf were ground to a powder and extracted using MeOH/H2O (80 : 20 v/v) at room temperature. The crude extract was then purified using aluminum oxide 150 basic, silica gel and (semi-)preparative high-performance liquid chromatography (HPLC). LOC, pachysiphine (PAC) and 3-oxotabersoine (OTAB) were isolated for the first time from the air-dried seeds of V. africana stapf, along with the known indole alkaloid tabersonine (TAB). NMR, HR-MS, FTIR, UV, melting point and optical rotation were utilized to elucidate the structures of LOC, PAC, OTAB and TAB, which were found to have slightly different spatial structures. Fluorescence spectrometry was used to study the interaction of LOC with BSA at different temperatures. This process revealed the quenching mechanism and measured the apparent constant (K). FTIR, Raman and CD were also employed to analyze the LOC–BSA interaction. In addition, the molecular docking of LOC to BSA was conducted using the CDOCKER docking protocol in Discovery Studio 3.1 (Accelrys Co. Ltd, USA) at the State Key Laboratory of Biotherapy (Sichuan University, China).
Experimental Preparation of stock solution BSA (essentially fatty-acid free), molecular mass assumed to be 66 500 Da, was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Stock solutions (20.0 μM) were prepared in Tris/HCl
* Correspondence to: Hui Li, College of Chemical Engineering, Sichuan University, Chengdu 610065, People’s Republic of China. E-mail: [email protected] College of Chemical Engineering, Sichuan University, Chengdu 610065, People’s Republic of China
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Figure 1. Molecular structure of lochnericine.
buffer pH 7.40, containing 0.10 M NaCl. Warfarin and ibuprofen were purchased from J&K Scientific Ltd. (Beijing, China). They were of analytical grade. LOC, OATB, PAC, TAB (HPLC ≥ 98%). Fluorescence experiments Fluorescence measurements were conducted on a Cary Eclipse fluorescence spectrophotometer (Varian, USA) equipped with 1.0 cm quartz cells. Based on preliminary experiments, the BSA concentration was kept at 2.0 μM, and the LOC concentration varied from 0 to 24.0 μM with a 4.0 μM gradient growth. The fluorescence spectra were measured using 5/5 nm (excitation/ emission) slit widths. The excitation wavelength was fixed at 280 nm, and the fluorescence spectra were recorded over the range of 300–500 nm at 294, 304 and 310 K. Interference experiments for similar components were performed by keeping the binary mixture of LOC–BSA in a 1 :1 ratio. In this study, all fluorescence intensities were corrected for the absorption of excited light and the reabsorption of emitted light based on the following relationship (23,24): Aex þAem
Fcorr ¼ Fobs e
2
(1)
where Fcorr and Fobs are the corrected and observed fluorescence intensities, respectively, and Aex and Aem are the absorption of the system at the excitation and emission wavelengths, respectively. FTIR, Raman and CD spectra measurements IR spectra were recorded on a Nicolet-6700 FTIR (Thermo, USA) spectrometer with a smart OMNI-sampler accessory. The FTIR spectra of BSA (0.2 mM) in the absence and presence of LOC (0.2 mM) were measured over the 4000–600 cm-1 range, with a resolution of 4 cm-1 and 64 scans at room temperature. The corresponding absorbance contributions of the buffer and free LOC solutions were recorded and digitally subtracted from the same instrumental parameters. The Raman spectra were measured with an in LabRAM HR (HORIBA, FRA), equipped with an argon ion laser excitation source with an emitting wavelength of 532 nm, and the LOC concentrations were set to 1.0 mM (ri = [LOC]/[BSA] = 0 : 1, 5 : 1). The CD spectra were recorded on a CD spectrometer (Model 400, AVIV, USA). CD measurements were recorded by keeping the concentration of BSA constant (2.0 μM) and varying the complex concentration from 0 to 16.00 μM (ri = [LOC]/[BSA] = 0 : 1, 8 : 1). Spectra were recorded at 298 K in a 2 mm path length quartz cell from 260 to 200 with a step size of 1 nm, bandwidth of 1 nm and an averaging time of 0.5 s.
structures of LOC were generated with ChemBioOffice 2010 and optimized with DS 3.1. For protein preparation, water was removed and the hydrogen atoms were added to pH 7.4 with DS 3.1. BSA was defined as a total receptor and a site sphere was built with a diameter of 5 Å based on 3,5diiodosalicylic acid (DIS). The pre-existing DIS was removed. From the receptor–ligand interaction section of DS 3.1, CDOCKER was chosen and all the default operating parameters were used unless pre-declared. During the docking process, freshly prepared LOC was used. CHARMm was selected as the force field. In this study, the heating steps were set as 2000 and the heating target temperature was 700. The cooling steps were set as 5000 with the cooling target temperature of 300. Because 3,5-diiodosalicylic acid has four binding sites in BSA (25), molecular docking occurred at each of these four sites. Ten molecular docking poses saved for each site were ranked according to -CDOCKER energy (–ECD). The pose with the highest –ECD value was chosen as the most suitable for the subsequent pose analysis.
Results and discussion Fluorescence quenching measurements The fluorescence intensity decreased significantly when the concentration of BSA was fixed at 2.0 μM at room temperature after the addition of LOC, as shown in Fig. 2. A combination effect between BSA and LOC occurred. This phenomenon was used to study the interactions between drugs and proteins (26). The red shifts suggested that the environment around tryptophan changed and was brought to a more hydrophilic condition (27). According to Geng’s study (28), the Stern–Volmer equation can be used to confirm the quenching mechanism by analyzing the fluorescence data at different temperatures. Higher temperatures result in faster diffusion and hence more collisional quenching. Higher temperatures typically result in the dissociation of weakly bound complexes, and hence smaller amounts of static quenching (29). Table 1 summarizes the calculated KSV
Molecular modeling The crystal structure of BSA was obtained from the Brookhaven Protein Data Bank (PDB ID: 4JK4). The LOC and the BSA protein were both pretreated initially. For ligand preparation, the 3D
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Figure 2. Fluorescence spectra of BSA in the presence of LOC at 294 K. The BSA concentration was 2 μM (A). The LOC concentration was 4 μM (B), 8 μM (C), 12 μM (D), 16 μM (E), 20 μM (F) and 24 μM (G). (Inset) Stern–Volmer curves for the binding of LOC with BSA at 294 K.
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Interaction between lochnericine and bovine serum albumin Table 1. Stern–Volmer quenching constants between LOC– BSA at different temperatures T (K)
KSV (×104 L/mol)
Ra
SDb
294 304 310
2.128 1.599 1.586
0.9996 0.9999 0.9967
0.0115 0.0089 0.0158
a
Correlation coefficient for the Ksv values. SD is the standard deviation.
b
at 294, 304 and 310 K. KSV decreased gradually with increasing temperature. A conclusion could be drawn: the fluorescence quenching between LOC and BSA was a static quenching procedure. Binding parameters Based on Anbazhagan and Naik’s study, Table 2 shows the static quenching constant (K) (30,31). The values of K suggested a medium binding force between LOC and BSA, indicating that LOC can be stored and carried by the protein in the body. However, drugs with excessively high affinity towards protein may impair the process of efficient distribution to the sites of the action and their elimination. This condition would then require correspondingly higher doses to achieve effective in vivo concentration (32). Binding mode The thermodynamic parameters of a binding reaction are mainly used to confirm the type of force governing the reaction (33). If the enthalpy change (ΔH°) over the temperature range under study is minimal, then the thermodynamic parameters ΔS° and ΔG° can be determined fro, the van’t Hoff equation (34). The results of the thermodynamic parameters are presented in Table 2. Because of the complex formation of LOC with BSA in aqueous solution, the positive value of ΔS° was regularly regarded as evidence of hydrophobic interaction; water molecules arranged in an orderly way around the ligand and protein acquired a more random configuration (35). By contrast, a negative ΔH° value was frequently taken as evidence for hydrogen bonds in the binding interaction (36). Conformational changes FTIR spectroscopy studies. Recently, FTIR has emerged as an efficient tool for the characterization of drug–protein interactions. The IR spectrum of a protein exhibits a number of amide bands,
representing different vibrations of the peptide moiety (37). Most investigations have concentrated on amide I band because it is sensitive to changes in the protein secondary structure (38,39). The component bands of amide I are distributed according to the well-established assignment criterion. The amide I peak occurs in the 1600–1700 cm-1 region (mainly the C = O stretch) and is widely used to investigate the secondary structure of proteins. In general, the band centered at ~ 1610–1640 cm-1 corresponds to β sheets, the band at ~ 1640–1650 cm-1 corresponds to random coils, the band at ~ 1650–1658 cm-1 corresponds to the α helix, and the band at ~ 1660–1700 cm-1 corresponds to the β turns. Quantitative analysis of the protein secondary structure of BSA before and after the interaction with LOC in Tris/HCl buffer pH 7.40 is shown in Fig. 3. With the addition of LOC, the peak at 1695 cm-1 (or 1674 cm-1) shifted to 1684 cm-1 (or 1672 cm-1), but the peak at 1632 cm-1 (or 1614 cm-1) shifted to 1636 cm-1 (or 1619 cm-1). The β-sheet content of BSA increased from 31.3 to 41.4%. The α-helix and β-turn content BSA decreased from 44.2 to 40.2% and from 24.5 to 18.4%, respectively. This indicates that the secondary structure of BSA changed from α helix and β turn to β sheets, and the loose nature of BSA is increased after addition of LOC. The observed spectral changes were due to major perturbations. These results indicated that LOC interacted with the C = O groups in the protein polypeptides. This phenomenon was believed to be due to unfolding of the protein as a result of the formation of H-bonds between BSA and LOC (40). The newly formed H-bonding was due to the electron density redistribution of the amide function group, which decreased the intensity of the original vibrations. The H-bonding affects the original bonding in α helix, β sheets and β turn, depending on the accessibility of the solvent and on the α-helix and β-sheet propensities of the protein. The LOC–BSA complexes caused rearrangement of the polypeptide carbonyl hydrogen-bonding network. Restrictions on the formation of hydrogen bonds in α helix and β turn explained the large effect on reducing the intensity percentage of them. Whereas the intrinsic β-sheet propensities were enhanced by the steric blocking effect (41). Raman spectroscopy studies. Raman spectra measurements were performed to obtain a deeper insight into the BSA conformation changes when it interacted with LOC. The spectrum of BSA changed greatly depends on binding. The amide I band in the 1610–1700 cm1 region was due to the Raman-active vibrational modes of the CONH peptidic bond, i.e. C = O stretching weakly coupled to inplane N–H bending (42,43). The curve fitting results were listed in Fig. 4. The results suggested that the α-helix content decreased from 53.3 to 50.6%, whereas the content of β sheet increased from 23.7 to 26.8%, the content of β turn decreased from 23.0 to 22.6%.
Table 2. Binding constants of the LOC-BSA system T (K)
K (×104 L/mol)
Ra
SDb
ΔG° (kJ/mol)
ΔH° (kJ/mol)
ΔS° (J/mol/K)
Rc
SDd
294 304 310
1.915 1.329 1.184
0.9996 0.9985 0.9972
0.0097 0.0143 0.0225
–24.10 –24.00 –24.17
–23.40
2.193
0.9907
0.0901
a
Correlation coefficient for the K values. SD is the standard deviation for the K values. c Correlation coefficient for the van’t Hoff plot. d SD is the standard deviation for the van’t Hoff plot. b
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–1
Figure 3. Second derivative resolution enhancement and curve-fitted amide I (1700–1600 cm ) region of free BSA (a) and LOC–BSA (b) in Tris/HCl buffer solution with CBSA = CLOC = 20 μM at pH 7.40.
Figure 4. The curve fitting of amide I of free BSA (a) and LOC–BSA (b).
CD spectroscopy studies. CD measurements were performed in the presence of different concentrations of LOC. It can be seen from Fig. 5 that BSA exhibited two negative ellipticities at 208 and 220 nm, which are characteristic of the α-helix structure of proteins (26). The shapes of the CD spectra were similar in the presence and absence of LOC, suggesting that the structure of BSA was also predominantly α helix. This indicated that LOC bound with the amino acid residues of the main polypeptide chain of protein and destroyed their hydrogen bonding
networks. Generally, the CD results were expressed in terms of mean residue ellipticity (MRE) in deg cm2/mol according to the following equation: MRE ¼
observedCDðm degÞ 10Cp nl
(2)
where Cp is the molar concentration of the protein, n is the number of amino acid residues (583 for BSA) and l is the path length of the cell. The α-helix content of free and combined BSA were calculated from MRE values at 208 nm using the following equation (10): α helixlð%Þ ¼
MRE208 4000 33000 4000
(3)
It can be calculated that the native BSA solution has a 58.4% α helix, whereas the α-helix content of BSA decreases to 55.9% on addition of LOC in a molar concentration ratio of 1 : 8. The decrease in the α-helix content indicated that the secondary structure of BSA changed during the reaction between BSA and LOC. This tendency (Table 3) was consistent with the above FTIR and Raman spectroscopy tendencies and further revealed that the LOC–BSA interactions affected the secondary and tertiary structures of BSA.
Figure 5. The CD spectra of of BSA in the presence of increasing amounts of LOC at pH 7.4. c(BSA) = 2.0 μM, the molar ratios of LOC to BSA were 0 : 1 (A) and 8 : 1(B), respectively.
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Interference experiment. Interference experiments were carried out with pachysiphine (PAC), tabersonine (TAB) and 3-oxotabersoine (OTAB). The percentage of the initial fluorescence due to the added ligand was calculated as:
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Interaction between lochnericine and bovine serum albumin Table 3. Secondary structure determination for free BSA and the LOC–BSA system in Tris/HCl buffer (pH 7.4) α-helix (%) β-sheet (%) β-turn (%)
System FTIR Raman CD
Free BSA LOC–BSA Free BSA LOC–BSA Free BSA LOC–BSA
44.2 40.2 53.3 50.6 58.4 55.9
31.3 41.4 23.7 26.8
24.5 18.4 23.0 22.6
ðfluorescence in presence of added ligandÞ= ðfluorescence in the absence of added ligandÞ 100% The changes induced by similar components are presented in Fig. 6. PAC, TAB and OTAB all competed with LOC, but the degrees of influence were different because of the slight difference in structure. There was only a minor difference in spatial structure between PAC and LOC. Hence, PAC competed more strongly with LOC compared with two other components. Limited by spatial steric hindrance, the competition ability of OTAB was relative weaker than TAB. Competition between the interfering component and LOC became more and more obvious with the addition interfering components. The structural distinction is the D ring, and this type of difference is found in the functional activity. In view of drug treatments for a particular disease, the traditional medical treatment used greater amounts of V. africana than LOC as a single drug because of the presence of competition. At the same time, increasing the traditional dose may increase the content of other components in blood and so increase side effects. Molecular modeling study. Most common drugs bind to sites I and II, located in subdomains IIA and IIIA of serum albumin. In Sekula’s study (25), BSA binds four molecules of DIS. Two DIS molecules were bound within the most active binding sites,
Figure 6. Effect of ligands on the fluorescence of BSA–LOC complexes. CBSA = CLOC = 2.0 μM, the molar ratios of ligands to LOC–BSA complexe were 2 : 1, 4 : 1, 6 : 1, 8 : 1, 10 : 1 and 12 : 1, respectively.
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Figure 7. The 2D docking mode of BSA with LOC in sites I (DS1).
drug-binding site 1 (DS1) and drug-binding site 2 (DS2) in subdomains IIA and IIIA, respectively. The third DIS molecule was bound in the cavity of subdomain IB (DS3). The DIS4-binding site (DS4) in BSA was located inside the DS1 cavity, close to the first DIS molecule, in the neighborhood of Trp213. In this study, LOC had 10 poses in DS1, DS2 and DS3, but no poses in DS4. The results showed that LOC could not only bind within sites I and II, but also within the cavity of subdomain IB. The 2D docking of BSA with LOC is presented in Figs 7, 8 and 9, respectively. LOC inserted well into those three sites. In Fig. 7, LOC is adjacent to hydrophobic residues such as Lys221, Leu237, Val240, Arg256, Ile289, Ala290 and Val292. In Fig. 8, LOC is adjacent to hydrophobic residues such as Ile327, Leu386, Leu429, Val432 and Leu452. In Fig. 9, LOC is adjacent to hydrophobic residues such as Leu115, Pro117, Leu122 and Phe164. At the same time, there were π → π* transitions between the A ring of LOC and Arg217. There also existed a hydrogen interaction between the C ring of LOC and Arg256, suggesting that the formation of hydrogen bonds decreased the hydrophobicity and increased the hydrophilicity to improve the stability of the
Figure 8. 2D docking of BSA with LOC in sites II (DS2).
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Figure 9. 2D docking of BSA with LOC in the cavity of subdomain IB (DS3).
LOC–BSA system. The arrow indicates the electron donor. The modeling results suggested that LOC interacted well with BSA, and the interaction between them was dominated by hydrophobic forces and hydrogen bonds, which is in agreement with the binding mode proposed by thermodynamic analysis. Furthermore, this result provided a good structural basis to explain the efficient fluorescence quenching of BSA emission in the presence of LOC.
Conclusions The interaction between LOC and BSA was studied by fluorescence spectroscopy under simulative physiological conditions. The experimental results showed that this interaction was a static quenching. The values of K suggested a medium binding force between LOC and BSA. The hydrophobic force and hydrogen bonds played major roles in the binding mode. This result was also confirmed by molecular modeling. Furthermore, FTIR, Raman and CD were used to investigate structural changes in BSA. The experimental results consistently indicated that LOC changed the secondary structure of BSA. At the same time, three structure-similar components were used in the interference experiments. The molecular modeling results showed that LOC could bind not only within sites I and II, but within the cavity of subdomain IB. In view of the standardized screens for pharmaceuticals this work would be useful in the design of new drugs and in clinical research. Analysis and research into the active components of the traditional drug are also necessary because of the presence of potential competition.
References 1. Bownik A. Harmful algae: effects of alkaloid cyanotoxins on animal and human health. Toxin Rev 2010;29:99–114. 2. Takahash N, Stresser DM, Williams DE, Bailey GS. Induction of hepatic CYP1A by indole-3-carbinol in protection against aflatoxin B1 hepatocarcinogenesis in rainbow trout. Fd Chem Toxic 1995; 33:841–50. 3. Park J-Y, Bjeldanes LF. Organ-selective induction of cytochrome P-450dependent activities by indole-3-carbinol-derived products: Influence on covalent binding of benzo[a]pyrene to hepatic and pulmonary DNA in the rat. Chem Biol Interact 1992;83:235–47.
wileyonlinelibrary.com/journal/luminescence
4. Koroch AR, Juliani HR, Kulakowski D, Arthur H, Asante-Dartey JE, Simon J. Voacanga africana: chemistry, quality and pharmacological activity. J Am Chem 2009;1021:363–80. 5. Tan PV, Penlap VB, Nyasse B, Nguemo JDB. Anti-ulcer actions of the bark methanol extract of Voacanga africana in different experimental ulcer models in rats. J Ethnopharmacol 2010;73:423–8. 6. Nair C, Pillay P. Lochnericine – a new alkaloid from Lochnera rosea. Tetrahedron 1959;6:89–91. 7. Moza B, Trojanek J, Bose AK, Das K, Funke P. The structure of lochnericine and lochnerinine. Tetrahedron Lett 1964;5:2561–6. 8. Yan J, Wang Q, Pan Q, Rao Z, Su Y, Li H. Assessment of the interaction between fraxinellone and bovine serum albumin by optical spectroscopy and molecular modeling methods. J Lumin 2013;137:180–5. 9. Wang Q, Yan J, He J, Bai K, Li H. Characterization of the interaction between 3-oxotabersonine and two serum albumins by using spectroscopic techniques. J Lumin 2013;138:1–7. 10. Du W, Teng T, Zhou C-C, Xi L, Wang J-Z. Spectroscopic studies on the interaction of bovine serum albumin with ginkgolic acid: binding characteristics and structural analysis. J Lumin 2012;132:1207–14. 11. Abdi K, Nafisi S, Manouchehri F, Bonsaii M, Khalaj A. Interaction of 5fluorouracil and its derivatives with bovine serum albumin. J Photochem Photobiol B 2012;107:20–6. 12. Bolel P, Mahapatra N, Halder M. Optical spectroscopic exploration of binding of Cochineal Red A with two homologous serum albumins. J Agric Food Chem 2012;60:3727–34. 13. He L, Wang X, Liu B, Wang J, Sun Y, Gao E, Xu S. Study on the interaction between promethazine hydrochloride and bovine serum albumin by fluorescence spectroscopy. J Lumin 2011;131:285–90. 14. Ma J, Wang L-Y, Xie M-X. Probing interaction of human serum albumin with the tautomers of 3,4′,7,8-tetrahydroxyflavone by spectroscopic and mass spectrometric approaches. J Photochem Photobiol A 2012;243:65–72. 15. Mote US, Bhattar SL, Patil SR, Kolekar GB. Interaction between felodipine and bovine serum albumin: fluorescence quenching study. Luminescence 2010;25:1–8. 16. Gómez AV, Ferrer EG, Añón MC, Puppo MC. Changes in secondary structure of gluten proteins due to emulsifiers. J Mol Struct 2013;1033:51–8. 17. Tian J, Xie Y, Zhao Y, Li C, Zhao S. Spectroscopy characterization of the interaction between brevifolin carboxylic acid and bovine serum albumin. Luminescence 2011;26:296–304. 18. Dong C, Ma S, Liu Y. Studies of the interaction between demeclocycline and human serum albumin by multi-spectroscopic and molecular docking methods. Spectrochim Acta A 2013;103:179–86. 19. Li J, Wang S. Application of molecular modelling and spectroscopic approaches for investigating the binding of tanshinone IIA to human serum albumin. J Chem Thermodynam 2013;58:206–10. 20. Xiao D, Zhang L, Wang Q, Lin X, Sun J, Li H. Investigations of the interactions of peimine and peiminine with human serum albumin by spectroscopic methods and docking studies. J Lumin 2014; 146:218–25. 21. Rodriguez Galdon B, Pinto Corraliza C, Cestero Carrillo JJ, Macias LP. Spectroscopic study of the interaction between lycopene and bovine serum albumin. Luminescence 2013;28:765–70. 22. Luo X, Du C, Wei J, Deng J, Lin Y, Lin C. Study of the interaction between sodium salts of (2E)-3-(4’-halophenyl)prop-2-enoyl sulfachloropyrazine and bovine serum albumin by fluorescence spectroscopy. Luminescence 2013;28:202–10. 23. Pinto Mdel C, Duque AL, Macias P. Fluorescence quenching study on the interaction between quercetin and lipoxygenase. J Fluoresc 2011;21:1311–18. 24. Shahabadi N, Mohammadpour M. Study on the interaction of sodium morin-5-sulfonate with bovine serum albumin by spectroscopic techniques. Spectrochim Acta A 2012;86:191–5. 25. Sekula B, Zielinski K, Bujacz A. Crystallographic studies of the complexes of bovine and equine serum albumin with 3,5-diiodosalicylic acid. Int J Biol Macromol 2013;60:316–24. 26. Suryawanshi VD, Anbhule PV, Gore AH, Patil SR, Kolekar GB. A spectral deciphering the perturbation of model transporter protein (HSA) by antibacterial pyrimidine derivative: pharmacokinetic and biophysical insights. J Photochem Photobiol B 2013,118:1–8. 27. Lakowicz JR. Principles of fluorescence spectroscopy. New York: Springer, 2009.
Copyright © 2014 John Wiley & Sons, Ltd.
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Interaction between lochnericine and bovine serum albumin 28. Geng S, Liu G, Li W, Cui F. Molecular interaction of ctDNA and HSA with sulfadiazine sodium by multispectroscopic methods and molecular modeling. Luminescence 2013;28:785–92. 29. Lakowicz JR, Weber G. Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale. Biochemistry 1973;12:4171–9. 30. Anbazhagan V, Renganathan R. Study on the binding of 2,3diazabicyclo[2.2.2]oct-2-ene with bovine serum albumin by fluorescence spectroscopy. J Lumin 2008;128:1454–8. 31. Xu X-W, Li X-J, Zhan S-H, Li Y-T, Wu Z-Y, Yan C-W. Synthesis and structure of a new tetracopper(II) complex bridged both by oxamido and phenolato groups: cytotoxic activity, and reactivity towards DNA and BSA. J Mol Struct 2013;1039:28–36. 32. Zhang J, Wang XJ, Yan YJ, Xiang WS. Comparative studies on the interaction of genistein, 8-chlorogenistein, and 3′,8-dichlorogenistein with bovine serum albumin. J Agric Food Chem 2011;59:7506–13. 33. Ross PD, Subramanian S. Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry 1981;20:3096–102. 34. Mortimer RG. Physical chemistry. Burlington, MA: Elsevier, 2008. 35. Li D, Zhu J, Jin J, Yao X. Studies on the binding of nevadensin to human serum albumin by molecular spectroscopy and modeling. J Mol Struct 2007;846:34–41.
Luminescence 2014
36. Jiang H, Chen R, Pu H. Study on the interaction between tabersonine and human serum albumin by optical spectroscopy and molecular modeling methods. J Lumin 2012;132:592–9. 37. David C, d’Andrea C, Lancelot E, Bochterle J, Guillot N, Fazio B, Maragò OM, Sutton A, Charnaux N, Neubrech F, Pucci A, Gucciardi PG, de la Chapelle ML. Raman and IR spectroscopy of manganese superoxide dismutase, a pathology biomarker. Vib Spectrosc 2012;62:50–8. 38. Hussein BHM. Spectroscopic studies of 7,8-dihydroxy-4-methylcoumarin and its interaction with bovine serum albumin. J Lumin 2011;131:900–8. 39. Tu S, Jiang X, Zhou L, Yin W, Wang H, Duan M, Liu P, Jiang X. Study of the interaction of gemini surfactant NAE12-4-12 with bovine serum albumin. J Lumin 2012;132:381–5. 40. Bai YW, Englander SW. Hydrogen bond strength and β-sheet propensities: the role of a side chain blocking effect. Protein Struct Funct Genet 1994;18:262–6. 41. Jackson M, Mantsch HH. The ise and misuse of FTIR spectroscopy in the determination of protein structure. Crit Rev Biochem Mol Biol 1995;30(2):95–120. 42. Peng X, Yu J, Yu Q, Bian H, Huang F, Liang H. Binding of engeletin with bovine serum albumin: insights from spectroscopic investigations. J Fluoresc 2012;22:511–19. 43. David C, Foley S, Mavon C, Enescu M. Reductive unfolding of serum albumins uncovered by Raman spectroscopy. Biopolymers 2008,89:623–34.
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Revised: 16 May 2014,
Accepted: 22 May 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2724
Spectroscopy and docking simulations of the interaction between lochnericine and bovine serum albumin Qing Wang, Jiawei He, Jin Yan, Di Wu and Hui Li* ABSTRACT: Lochnericine (LOC) is a component of Voacanga africana, which is a type of traditional medical food in Africa widely used for treating diseases. In this article, the interaction between LOC and bovine serum albumin (BSA) was studied by fluorescence spectroscopy. Furthermore, Fourier transform infrared (FTIR), Raman and circular dichroism (CD) were used to investigate the structural changes of BSA. The experimental results consistently indicated that LOC changed the secondary structure of BSA. Three structure-similar components were used to study the interference experiments. The molecular modeling results showed that LOC could bind within not only sites I and II, but also bind the cavity of subdomain IB. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: lochnericine; bovine serum albumin (BSA); interaction; structural changes; molecular modeling
Introduction Alkaloids are a group of naturally occurring chemical compounds that mostly contain basic nitrogen atoms, and have diverse and important physiological effects on humans and other animals (1). Therefore, alkaloids have been the subject of much clinical research, and traditional medical foods rich in alkaloids have attracted interest in recent years (2,3). Alkaloids are the main active components of Voacanga africana (4). In Africa, V. africana is used to treat diseases like leprosy, diarrhea, generalized edema, convulsions in children, orchitis and ectopic testes gonorrhea (5). Lochnericine (LOC), which is a type of indole alkaloid, can be extracted from V. africana. Although LOC was discovered long ago and its structure (Fig. 1) has been identified previously (6,7), it has not been used as a single drug. Interactions between potential medical LOC and serum albumin must be considered because of the relevance to its transport, biological activity and clearance. Serum albumin, the most abundant protein present in the circulatory system of animals, is a major macromolecule that contributes to osmotic blood pressure. Serum albumin can bind various compounds (8,9). When drugs are absorbed into the circulatory system, they can extensively and reversibly bind to serum albumin. The interaction between drug and protein can significantly affect the biological activity and toxicity of drugs in pharmacology (10–12). Usually, the activity of a protein is closely related to its conformation and structure, and gives information critical to understanding the relationship between structure and function (13,14). Thus, the identification of a protein through its conformational features is essential in the medical applications (15). Fourier transform infrared (FTIR) spectroscopy, Raman scattering and circular dichroism (CD) spectroscopy are powerful tools for investigating structural changes in serum albumin (16–19). They provide important information on both the secondary and tertiary structures of proteins, as well as on the protein microenvironment (20). Bovine serum albumin (BSA) was selected as the model protein in this work due to its abundance, low cost, ease of purification and
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stability. The results of all the BSA studies were consistent with the fact that human and bovine serum albumins are homologous proteins (21,22). In this work, air-dried seeds of V. africana stapf were ground to a powder and extracted using MeOH/H2O (80 : 20 v/v) at room temperature. The crude extract was then purified using aluminum oxide 150 basic, silica gel and (semi-)preparative high-performance liquid chromatography (HPLC). LOC, pachysiphine (PAC) and 3-oxotabersoine (OTAB) were isolated for the first time from the air-dried seeds of V. africana stapf, along with the known indole alkaloid tabersonine (TAB). NMR, HR-MS, FTIR, UV, melting point and optical rotation were utilized to elucidate the structures of LOC, PAC, OTAB and TAB, which were found to have slightly different spatial structures. Fluorescence spectrometry was used to study the interaction of LOC with BSA at different temperatures. This process revealed the quenching mechanism and measured the apparent constant (K). FTIR, Raman and CD were also employed to analyze the LOC–BSA interaction. In addition, the molecular docking of LOC to BSA was conducted using the CDOCKER docking protocol in Discovery Studio 3.1 (Accelrys Co. Ltd, USA) at the State Key Laboratory of Biotherapy (Sichuan University, China).
Experimental Preparation of stock solution BSA (essentially fatty-acid free), molecular mass assumed to be 66 500 Da, was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Stock solutions (20.0 μM) were prepared in Tris/HCl
* Correspondence to: Hui Li, College of Chemical Engineering, Sichuan University, Chengdu 610065, People’s Republic of China. E-mail: [email protected] College of Chemical Engineering, Sichuan University, Chengdu 610065, People’s Republic of China
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Figure 1. Molecular structure of lochnericine.
buffer pH 7.40, containing 0.10 M NaCl. Warfarin and ibuprofen were purchased from J&K Scientific Ltd. (Beijing, China). They were of analytical grade. LOC, OATB, PAC, TAB (HPLC ≥ 98%). Fluorescence experiments Fluorescence measurements were conducted on a Cary Eclipse fluorescence spectrophotometer (Varian, USA) equipped with 1.0 cm quartz cells. Based on preliminary experiments, the BSA concentration was kept at 2.0 μM, and the LOC concentration varied from 0 to 24.0 μM with a 4.0 μM gradient growth. The fluorescence spectra were measured using 5/5 nm (excitation/ emission) slit widths. The excitation wavelength was fixed at 280 nm, and the fluorescence spectra were recorded over the range of 300–500 nm at 294, 304 and 310 K. Interference experiments for similar components were performed by keeping the binary mixture of LOC–BSA in a 1 :1 ratio. In this study, all fluorescence intensities were corrected for the absorption of excited light and the reabsorption of emitted light based on the following relationship (23,24): Aex þAem
Fcorr ¼ Fobs e
2
(1)
where Fcorr and Fobs are the corrected and observed fluorescence intensities, respectively, and Aex and Aem are the absorption of the system at the excitation and emission wavelengths, respectively. FTIR, Raman and CD spectra measurements IR spectra were recorded on a Nicolet-6700 FTIR (Thermo, USA) spectrometer with a smart OMNI-sampler accessory. The FTIR spectra of BSA (0.2 mM) in the absence and presence of LOC (0.2 mM) were measured over the 4000–600 cm-1 range, with a resolution of 4 cm-1 and 64 scans at room temperature. The corresponding absorbance contributions of the buffer and free LOC solutions were recorded and digitally subtracted from the same instrumental parameters. The Raman spectra were measured with an in LabRAM HR (HORIBA, FRA), equipped with an argon ion laser excitation source with an emitting wavelength of 532 nm, and the LOC concentrations were set to 1.0 mM (ri = [LOC]/[BSA] = 0 : 1, 5 : 1). The CD spectra were recorded on a CD spectrometer (Model 400, AVIV, USA). CD measurements were recorded by keeping the concentration of BSA constant (2.0 μM) and varying the complex concentration from 0 to 16.00 μM (ri = [LOC]/[BSA] = 0 : 1, 8 : 1). Spectra were recorded at 298 K in a 2 mm path length quartz cell from 260 to 200 with a step size of 1 nm, bandwidth of 1 nm and an averaging time of 0.5 s.
structures of LOC were generated with ChemBioOffice 2010 and optimized with DS 3.1. For protein preparation, water was removed and the hydrogen atoms were added to pH 7.4 with DS 3.1. BSA was defined as a total receptor and a site sphere was built with a diameter of 5 Å based on 3,5diiodosalicylic acid (DIS). The pre-existing DIS was removed. From the receptor–ligand interaction section of DS 3.1, CDOCKER was chosen and all the default operating parameters were used unless pre-declared. During the docking process, freshly prepared LOC was used. CHARMm was selected as the force field. In this study, the heating steps were set as 2000 and the heating target temperature was 700. The cooling steps were set as 5000 with the cooling target temperature of 300. Because 3,5-diiodosalicylic acid has four binding sites in BSA (25), molecular docking occurred at each of these four sites. Ten molecular docking poses saved for each site were ranked according to -CDOCKER energy (–ECD). The pose with the highest –ECD value was chosen as the most suitable for the subsequent pose analysis.
Results and discussion Fluorescence quenching measurements The fluorescence intensity decreased significantly when the concentration of BSA was fixed at 2.0 μM at room temperature after the addition of LOC, as shown in Fig. 2. A combination effect between BSA and LOC occurred. This phenomenon was used to study the interactions between drugs and proteins (26). The red shifts suggested that the environment around tryptophan changed and was brought to a more hydrophilic condition (27). According to Geng’s study (28), the Stern–Volmer equation can be used to confirm the quenching mechanism by analyzing the fluorescence data at different temperatures. Higher temperatures result in faster diffusion and hence more collisional quenching. Higher temperatures typically result in the dissociation of weakly bound complexes, and hence smaller amounts of static quenching (29). Table 1 summarizes the calculated KSV
Molecular modeling The crystal structure of BSA was obtained from the Brookhaven Protein Data Bank (PDB ID: 4JK4). The LOC and the BSA protein were both pretreated initially. For ligand preparation, the 3D
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Figure 2. Fluorescence spectra of BSA in the presence of LOC at 294 K. The BSA concentration was 2 μM (A). The LOC concentration was 4 μM (B), 8 μM (C), 12 μM (D), 16 μM (E), 20 μM (F) and 24 μM (G). (Inset) Stern–Volmer curves for the binding of LOC with BSA at 294 K.
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Interaction between lochnericine and bovine serum albumin Table 1. Stern–Volmer quenching constants between LOC– BSA at different temperatures T (K)
KSV (×104 L/mol)
Ra
SDb
294 304 310
2.128 1.599 1.586
0.9996 0.9999 0.9967
0.0115 0.0089 0.0158
a
Correlation coefficient for the Ksv values. SD is the standard deviation.
b
at 294, 304 and 310 K. KSV decreased gradually with increasing temperature. A conclusion could be drawn: the fluorescence quenching between LOC and BSA was a static quenching procedure. Binding parameters Based on Anbazhagan and Naik’s study, Table 2 shows the static quenching constant (K) (30,31). The values of K suggested a medium binding force between LOC and BSA, indicating that LOC can be stored and carried by the protein in the body. However, drugs with excessively high affinity towards protein may impair the process of efficient distribution to the sites of the action and their elimination. This condition would then require correspondingly higher doses to achieve effective in vivo concentration (32). Binding mode The thermodynamic parameters of a binding reaction are mainly used to confirm the type of force governing the reaction (33). If the enthalpy change (ΔH°) over the temperature range under study is minimal, then the thermodynamic parameters ΔS° and ΔG° can be determined fro, the van’t Hoff equation (34). The results of the thermodynamic parameters are presented in Table 2. Because of the complex formation of LOC with BSA in aqueous solution, the positive value of ΔS° was regularly regarded as evidence of hydrophobic interaction; water molecules arranged in an orderly way around the ligand and protein acquired a more random configuration (35). By contrast, a negative ΔH° value was frequently taken as evidence for hydrogen bonds in the binding interaction (36). Conformational changes FTIR spectroscopy studies. Recently, FTIR has emerged as an efficient tool for the characterization of drug–protein interactions. The IR spectrum of a protein exhibits a number of amide bands,
representing different vibrations of the peptide moiety (37). Most investigations have concentrated on amide I band because it is sensitive to changes in the protein secondary structure (38,39). The component bands of amide I are distributed according to the well-established assignment criterion. The amide I peak occurs in the 1600–1700 cm-1 region (mainly the C = O stretch) and is widely used to investigate the secondary structure of proteins. In general, the band centered at ~ 1610–1640 cm-1 corresponds to β sheets, the band at ~ 1640–1650 cm-1 corresponds to random coils, the band at ~ 1650–1658 cm-1 corresponds to the α helix, and the band at ~ 1660–1700 cm-1 corresponds to the β turns. Quantitative analysis of the protein secondary structure of BSA before and after the interaction with LOC in Tris/HCl buffer pH 7.40 is shown in Fig. 3. With the addition of LOC, the peak at 1695 cm-1 (or 1674 cm-1) shifted to 1684 cm-1 (or 1672 cm-1), but the peak at 1632 cm-1 (or 1614 cm-1) shifted to 1636 cm-1 (or 1619 cm-1). The β-sheet content of BSA increased from 31.3 to 41.4%. The α-helix and β-turn content BSA decreased from 44.2 to 40.2% and from 24.5 to 18.4%, respectively. This indicates that the secondary structure of BSA changed from α helix and β turn to β sheets, and the loose nature of BSA is increased after addition of LOC. The observed spectral changes were due to major perturbations. These results indicated that LOC interacted with the C = O groups in the protein polypeptides. This phenomenon was believed to be due to unfolding of the protein as a result of the formation of H-bonds between BSA and LOC (40). The newly formed H-bonding was due to the electron density redistribution of the amide function group, which decreased the intensity of the original vibrations. The H-bonding affects the original bonding in α helix, β sheets and β turn, depending on the accessibility of the solvent and on the α-helix and β-sheet propensities of the protein. The LOC–BSA complexes caused rearrangement of the polypeptide carbonyl hydrogen-bonding network. Restrictions on the formation of hydrogen bonds in α helix and β turn explained the large effect on reducing the intensity percentage of them. Whereas the intrinsic β-sheet propensities were enhanced by the steric blocking effect (41). Raman spectroscopy studies. Raman spectra measurements were performed to obtain a deeper insight into the BSA conformation changes when it interacted with LOC. The spectrum of BSA changed greatly depends on binding. The amide I band in the 1610–1700 cm1 region was due to the Raman-active vibrational modes of the CONH peptidic bond, i.e. C = O stretching weakly coupled to inplane N–H bending (42,43). The curve fitting results were listed in Fig. 4. The results suggested that the α-helix content decreased from 53.3 to 50.6%, whereas the content of β sheet increased from 23.7 to 26.8%, the content of β turn decreased from 23.0 to 22.6%.
Table 2. Binding constants of the LOC-BSA system T (K)
K (×104 L/mol)
Ra
SDb
ΔG° (kJ/mol)
ΔH° (kJ/mol)
ΔS° (J/mol/K)
Rc
SDd
294 304 310
1.915 1.329 1.184
0.9996 0.9985 0.9972
0.0097 0.0143 0.0225
–24.10 –24.00 –24.17
–23.40
2.193
0.9907
0.0901
a
Correlation coefficient for the K values. SD is the standard deviation for the K values. c Correlation coefficient for the van’t Hoff plot. d SD is the standard deviation for the van’t Hoff plot. b
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–1
Figure 3. Second derivative resolution enhancement and curve-fitted amide I (1700–1600 cm ) region of free BSA (a) and LOC–BSA (b) in Tris/HCl buffer solution with CBSA = CLOC = 20 μM at pH 7.40.
Figure 4. The curve fitting of amide I of free BSA (a) and LOC–BSA (b).
CD spectroscopy studies. CD measurements were performed in the presence of different concentrations of LOC. It can be seen from Fig. 5 that BSA exhibited two negative ellipticities at 208 and 220 nm, which are characteristic of the α-helix structure of proteins (26). The shapes of the CD spectra were similar in the presence and absence of LOC, suggesting that the structure of BSA was also predominantly α helix. This indicated that LOC bound with the amino acid residues of the main polypeptide chain of protein and destroyed their hydrogen bonding
networks. Generally, the CD results were expressed in terms of mean residue ellipticity (MRE) in deg cm2/mol according to the following equation: MRE ¼
observedCDðm degÞ 10Cp nl
(2)
where Cp is the molar concentration of the protein, n is the number of amino acid residues (583 for BSA) and l is the path length of the cell. The α-helix content of free and combined BSA were calculated from MRE values at 208 nm using the following equation (10): α helixlð%Þ ¼
MRE208 4000 33000 4000
(3)
It can be calculated that the native BSA solution has a 58.4% α helix, whereas the α-helix content of BSA decreases to 55.9% on addition of LOC in a molar concentration ratio of 1 : 8. The decrease in the α-helix content indicated that the secondary structure of BSA changed during the reaction between BSA and LOC. This tendency (Table 3) was consistent with the above FTIR and Raman spectroscopy tendencies and further revealed that the LOC–BSA interactions affected the secondary and tertiary structures of BSA.
Figure 5. The CD spectra of of BSA in the presence of increasing amounts of LOC at pH 7.4. c(BSA) = 2.0 μM, the molar ratios of LOC to BSA were 0 : 1 (A) and 8 : 1(B), respectively.
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Interference experiment. Interference experiments were carried out with pachysiphine (PAC), tabersonine (TAB) and 3-oxotabersoine (OTAB). The percentage of the initial fluorescence due to the added ligand was calculated as:
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Interaction between lochnericine and bovine serum albumin Table 3. Secondary structure determination for free BSA and the LOC–BSA system in Tris/HCl buffer (pH 7.4) α-helix (%) β-sheet (%) β-turn (%)
System FTIR Raman CD
Free BSA LOC–BSA Free BSA LOC–BSA Free BSA LOC–BSA
44.2 40.2 53.3 50.6 58.4 55.9
31.3 41.4 23.7 26.8
24.5 18.4 23.0 22.6
ðfluorescence in presence of added ligandÞ= ðfluorescence in the absence of added ligandÞ 100% The changes induced by similar components are presented in Fig. 6. PAC, TAB and OTAB all competed with LOC, but the degrees of influence were different because of the slight difference in structure. There was only a minor difference in spatial structure between PAC and LOC. Hence, PAC competed more strongly with LOC compared with two other components. Limited by spatial steric hindrance, the competition ability of OTAB was relative weaker than TAB. Competition between the interfering component and LOC became more and more obvious with the addition interfering components. The structural distinction is the D ring, and this type of difference is found in the functional activity. In view of drug treatments for a particular disease, the traditional medical treatment used greater amounts of V. africana than LOC as a single drug because of the presence of competition. At the same time, increasing the traditional dose may increase the content of other components in blood and so increase side effects. Molecular modeling study. Most common drugs bind to sites I and II, located in subdomains IIA and IIIA of serum albumin. In Sekula’s study (25), BSA binds four molecules of DIS. Two DIS molecules were bound within the most active binding sites,
Figure 6. Effect of ligands on the fluorescence of BSA–LOC complexes. CBSA = CLOC = 2.0 μM, the molar ratios of ligands to LOC–BSA complexe were 2 : 1, 4 : 1, 6 : 1, 8 : 1, 10 : 1 and 12 : 1, respectively.
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Figure 7. The 2D docking mode of BSA with LOC in sites I (DS1).
drug-binding site 1 (DS1) and drug-binding site 2 (DS2) in subdomains IIA and IIIA, respectively. The third DIS molecule was bound in the cavity of subdomain IB (DS3). The DIS4-binding site (DS4) in BSA was located inside the DS1 cavity, close to the first DIS molecule, in the neighborhood of Trp213. In this study, LOC had 10 poses in DS1, DS2 and DS3, but no poses in DS4. The results showed that LOC could not only bind within sites I and II, but also within the cavity of subdomain IB. The 2D docking of BSA with LOC is presented in Figs 7, 8 and 9, respectively. LOC inserted well into those three sites. In Fig. 7, LOC is adjacent to hydrophobic residues such as Lys221, Leu237, Val240, Arg256, Ile289, Ala290 and Val292. In Fig. 8, LOC is adjacent to hydrophobic residues such as Ile327, Leu386, Leu429, Val432 and Leu452. In Fig. 9, LOC is adjacent to hydrophobic residues such as Leu115, Pro117, Leu122 and Phe164. At the same time, there were π → π* transitions between the A ring of LOC and Arg217. There also existed a hydrogen interaction between the C ring of LOC and Arg256, suggesting that the formation of hydrogen bonds decreased the hydrophobicity and increased the hydrophilicity to improve the stability of the
Figure 8. 2D docking of BSA with LOC in sites II (DS2).
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Figure 9. 2D docking of BSA with LOC in the cavity of subdomain IB (DS3).
LOC–BSA system. The arrow indicates the electron donor. The modeling results suggested that LOC interacted well with BSA, and the interaction between them was dominated by hydrophobic forces and hydrogen bonds, which is in agreement with the binding mode proposed by thermodynamic analysis. Furthermore, this result provided a good structural basis to explain the efficient fluorescence quenching of BSA emission in the presence of LOC.
Conclusions The interaction between LOC and BSA was studied by fluorescence spectroscopy under simulative physiological conditions. The experimental results showed that this interaction was a static quenching. The values of K suggested a medium binding force between LOC and BSA. The hydrophobic force and hydrogen bonds played major roles in the binding mode. This result was also confirmed by molecular modeling. Furthermore, FTIR, Raman and CD were used to investigate structural changes in BSA. The experimental results consistently indicated that LOC changed the secondary structure of BSA. At the same time, three structure-similar components were used in the interference experiments. The molecular modeling results showed that LOC could bind not only within sites I and II, but within the cavity of subdomain IB. In view of the standardized screens for pharmaceuticals this work would be useful in the design of new drugs and in clinical research. Analysis and research into the active components of the traditional drug are also necessary because of the presence of potential competition.
References 1. Bownik A. Harmful algae: effects of alkaloid cyanotoxins on animal and human health. Toxin Rev 2010;29:99–114. 2. Takahash N, Stresser DM, Williams DE, Bailey GS. Induction of hepatic CYP1A by indole-3-carbinol in protection against aflatoxin B1 hepatocarcinogenesis in rainbow trout. Fd Chem Toxic 1995; 33:841–50. 3. Park J-Y, Bjeldanes LF. Organ-selective induction of cytochrome P-450dependent activities by indole-3-carbinol-derived products: Influence on covalent binding of benzo[a]pyrene to hepatic and pulmonary DNA in the rat. Chem Biol Interact 1992;83:235–47.
wileyonlinelibrary.com/journal/luminescence
4. Koroch AR, Juliani HR, Kulakowski D, Arthur H, Asante-Dartey JE, Simon J. Voacanga africana: chemistry, quality and pharmacological activity. J Am Chem 2009;1021:363–80. 5. Tan PV, Penlap VB, Nyasse B, Nguemo JDB. Anti-ulcer actions of the bark methanol extract of Voacanga africana in different experimental ulcer models in rats. J Ethnopharmacol 2010;73:423–8. 6. Nair C, Pillay P. Lochnericine – a new alkaloid from Lochnera rosea. Tetrahedron 1959;6:89–91. 7. Moza B, Trojanek J, Bose AK, Das K, Funke P. The structure of lochnericine and lochnerinine. Tetrahedron Lett 1964;5:2561–6. 8. Yan J, Wang Q, Pan Q, Rao Z, Su Y, Li H. Assessment of the interaction between fraxinellone and bovine serum albumin by optical spectroscopy and molecular modeling methods. J Lumin 2013;137:180–5. 9. Wang Q, Yan J, He J, Bai K, Li H. Characterization of the interaction between 3-oxotabersonine and two serum albumins by using spectroscopic techniques. J Lumin 2013;138:1–7. 10. Du W, Teng T, Zhou C-C, Xi L, Wang J-Z. Spectroscopic studies on the interaction of bovine serum albumin with ginkgolic acid: binding characteristics and structural analysis. J Lumin 2012;132:1207–14. 11. Abdi K, Nafisi S, Manouchehri F, Bonsaii M, Khalaj A. Interaction of 5fluorouracil and its derivatives with bovine serum albumin. J Photochem Photobiol B 2012;107:20–6. 12. Bolel P, Mahapatra N, Halder M. Optical spectroscopic exploration of binding of Cochineal Red A with two homologous serum albumins. J Agric Food Chem 2012;60:3727–34. 13. He L, Wang X, Liu B, Wang J, Sun Y, Gao E, Xu S. Study on the interaction between promethazine hydrochloride and bovine serum albumin by fluorescence spectroscopy. J Lumin 2011;131:285–90. 14. Ma J, Wang L-Y, Xie M-X. Probing interaction of human serum albumin with the tautomers of 3,4′,7,8-tetrahydroxyflavone by spectroscopic and mass spectrometric approaches. J Photochem Photobiol A 2012;243:65–72. 15. Mote US, Bhattar SL, Patil SR, Kolekar GB. Interaction between felodipine and bovine serum albumin: fluorescence quenching study. Luminescence 2010;25:1–8. 16. Gómez AV, Ferrer EG, Añón MC, Puppo MC. Changes in secondary structure of gluten proteins due to emulsifiers. J Mol Struct 2013;1033:51–8. 17. Tian J, Xie Y, Zhao Y, Li C, Zhao S. Spectroscopy characterization of the interaction between brevifolin carboxylic acid and bovine serum albumin. Luminescence 2011;26:296–304. 18. Dong C, Ma S, Liu Y. Studies of the interaction between demeclocycline and human serum albumin by multi-spectroscopic and molecular docking methods. Spectrochim Acta A 2013;103:179–86. 19. Li J, Wang S. Application of molecular modelling and spectroscopic approaches for investigating the binding of tanshinone IIA to human serum albumin. J Chem Thermodynam 2013;58:206–10. 20. Xiao D, Zhang L, Wang Q, Lin X, Sun J, Li H. Investigations of the interactions of peimine and peiminine with human serum albumin by spectroscopic methods and docking studies. J Lumin 2014; 146:218–25. 21. Rodriguez Galdon B, Pinto Corraliza C, Cestero Carrillo JJ, Macias LP. Spectroscopic study of the interaction between lycopene and bovine serum albumin. Luminescence 2013;28:765–70. 22. Luo X, Du C, Wei J, Deng J, Lin Y, Lin C. Study of the interaction between sodium salts of (2E)-3-(4’-halophenyl)prop-2-enoyl sulfachloropyrazine and bovine serum albumin by fluorescence spectroscopy. Luminescence 2013;28:202–10. 23. Pinto Mdel C, Duque AL, Macias P. Fluorescence quenching study on the interaction between quercetin and lipoxygenase. J Fluoresc 2011;21:1311–18. 24. Shahabadi N, Mohammadpour M. Study on the interaction of sodium morin-5-sulfonate with bovine serum albumin by spectroscopic techniques. Spectrochim Acta A 2012;86:191–5. 25. Sekula B, Zielinski K, Bujacz A. Crystallographic studies of the complexes of bovine and equine serum albumin with 3,5-diiodosalicylic acid. Int J Biol Macromol 2013;60:316–24. 26. Suryawanshi VD, Anbhule PV, Gore AH, Patil SR, Kolekar GB. A spectral deciphering the perturbation of model transporter protein (HSA) by antibacterial pyrimidine derivative: pharmacokinetic and biophysical insights. J Photochem Photobiol B 2013,118:1–8. 27. Lakowicz JR. Principles of fluorescence spectroscopy. New York: Springer, 2009.
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014
Interaction between lochnericine and bovine serum albumin 28. Geng S, Liu G, Li W, Cui F. Molecular interaction of ctDNA and HSA with sulfadiazine sodium by multispectroscopic methods and molecular modeling. Luminescence 2013;28:785–92. 29. Lakowicz JR, Weber G. Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale. Biochemistry 1973;12:4171–9. 30. Anbazhagan V, Renganathan R. Study on the binding of 2,3diazabicyclo[2.2.2]oct-2-ene with bovine serum albumin by fluorescence spectroscopy. J Lumin 2008;128:1454–8. 31. Xu X-W, Li X-J, Zhan S-H, Li Y-T, Wu Z-Y, Yan C-W. Synthesis and structure of a new tetracopper(II) complex bridged both by oxamido and phenolato groups: cytotoxic activity, and reactivity towards DNA and BSA. J Mol Struct 2013;1039:28–36. 32. Zhang J, Wang XJ, Yan YJ, Xiang WS. Comparative studies on the interaction of genistein, 8-chlorogenistein, and 3′,8-dichlorogenistein with bovine serum albumin. J Agric Food Chem 2011;59:7506–13. 33. Ross PD, Subramanian S. Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry 1981;20:3096–102. 34. Mortimer RG. Physical chemistry. Burlington, MA: Elsevier, 2008. 35. Li D, Zhu J, Jin J, Yao X. Studies on the binding of nevadensin to human serum albumin by molecular spectroscopy and modeling. J Mol Struct 2007;846:34–41.
Luminescence 2014
36. Jiang H, Chen R, Pu H. Study on the interaction between tabersonine and human serum albumin by optical spectroscopy and molecular modeling methods. J Lumin 2012;132:592–9. 37. David C, d’Andrea C, Lancelot E, Bochterle J, Guillot N, Fazio B, Maragò OM, Sutton A, Charnaux N, Neubrech F, Pucci A, Gucciardi PG, de la Chapelle ML. Raman and IR spectroscopy of manganese superoxide dismutase, a pathology biomarker. Vib Spectrosc 2012;62:50–8. 38. Hussein BHM. Spectroscopic studies of 7,8-dihydroxy-4-methylcoumarin and its interaction with bovine serum albumin. J Lumin 2011;131:900–8. 39. Tu S, Jiang X, Zhou L, Yin W, Wang H, Duan M, Liu P, Jiang X. Study of the interaction of gemini surfactant NAE12-4-12 with bovine serum albumin. J Lumin 2012;132:381–5. 40. Bai YW, Englander SW. Hydrogen bond strength and β-sheet propensities: the role of a side chain blocking effect. Protein Struct Funct Genet 1994;18:262–6. 41. Jackson M, Mantsch HH. The ise and misuse of FTIR spectroscopy in the determination of protein structure. Crit Rev Biochem Mol Biol 1995;30(2):95–120. 42. Peng X, Yu J, Yu Q, Bian H, Huang F, Liang H. Binding of engeletin with bovine serum albumin: insights from spectroscopic investigations. J Fluoresc 2012;22:511–19. 43. David C, Foley S, Mavon C, Enescu M. Reductive unfolding of serum albumins uncovered by Raman spectroscopy. Biopolymers 2008,89:623–34.
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