Hindawi Publishing Corporation Journal of Chemistry Volume 2013, Article ID 308054, 6 pages http://dx.doi.org/10.1155/2013/308054

Research Article Fluorescence Spectroscopy Study on the Interaction between Evodiamine and Bovine Serum Albumin

Mingxiong Tan,1, 2 Weijiang Liang,1 Xujian Luo,1 and Yunqiong Gu1

1 School of Chemistry and Material, Yulin Normal University, Yulin 537000, 2 e Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry & Chemical Engineering, Normal University, Guilin 541004, China

Correspondence should be addressed to Mingxiong Tan; [email protected]

Received 14 June 2012; Accepted 3 December 2012

Academic Editor: Tomokazu Yoshimura

Copyright © 2013 Mingxiong Tan et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

e interaction of evodiamine (Evo) with bovine serum albumins (BSAs) at different two temperatures (298 and 310 K) was investigated by means of �uorescence spectroscopy. e experimental results showed that Evo binds with BSA via a static quenching procedure with association constants of L/mol at 298 K and L/mol at 310 K. e number of bound Evo molecules per protein is 1.31 at 298 K and 1.33 at 310 K.6 e results suggested that Evo5 reacts with BSA chie�y through hydrophobic and electrostatic interactions, and it does𝐾𝐾 not1.61 alter × the 10 -helical nature of BAS.6.78 × 10

𝛼𝛼 1. Introduction Serum albumin (SA) is a multiple function protein and acts as the transporter and disposition of many endogenous Evodiamine (Figure 1), a quinolone alkaloid, is the major and exogenous ligands, including fatty acids, amino acids, component isolated from the fruit of Evodia rutaecarpa, metals ions, and numerous pharmaceuticals by means of which is a traditional medicinal plant distributed in East hydrogen bonding, hydrophobic, electrostatic, and metal Asia, especially in China, and has been used for a long interactions [4–7]. e interaction intensity between anti- time as a traditional Chinese medicine for the treatment of tumor drugs and SA may effect on their bioavailability gastrointestinal disorders, headache, and postpartum hem- and toxicity [8–10]. In this regard, bovine serum albumin orrhage. In pharmacology studies, it has been reported that (BSA) has been studied extensively, partly because of its evodiamine (Evo) was found to have antinociceptive, anti- in�ammatory, antiobesity, vasodilatory, thermoregulatory, structural homology with human serum albumin (HSA). analgesic, cardiotonic, uterotonic, and antitumor activities BSA is composed of three linearly arranged, structurally [1–3]. Studies concerning screening of alkaloids have shown homologous subdomains. It has two tryptophan residues that that evodiamine exhibits the strongest cytotoxicity activity possess intrinsic domains, and each domain in turn is the against human colon and hepatoblastoma cell lines and product of two �uorescence: Trp134, which is located on the inhibitory activity on human colon carcinoma cell. Further surface of subdomain IB, and Trp212, locating within the studies demonstrate that evodiamine has antitumor potential hydrophobic binding pocket of subdomain IIA. e binding by inhibiting proliferation, inducing apoptosis and reducing sites of BSA for endogenous and exogenous ligands may be in invasion and metastasis of a wide variety of tumor cells, these domains. including breast cancer cells, prostate cancer cells, leukemic Fluorescence quenching is considered as a method for T-lymphocyte cells, melanoma cells, cervical cancer cells, measuring binding affinities. Fluorescence quenching is the colon cancer cells, and lung cancer cells. More importantly, decrease of the quantum yield of �uorescence from a �uo- evodiamine not only sensitizes chemoresistant breast cancer rophore induced by a variety of molecular interactions with cells to adriamycin, but also shows little toxicity against quencher molecule [11, 12]. It is, therefore, of interest to use normal human peripheral blood cells [4]. quenching of the intrinsic tryptophan �uorescence of BSA 2 Journal of Chemistry

O

N H N

H3C

F 1: e structure of Evodiamine.

(Trp-212 and Trp-134) as a tool to study the interaction of So, Evo does not obviously affect the -helical conformation BSA and evodiamine (Evo) [13, 14]. of BSA. 𝛼𝛼 2. Results and Discussion 2.2. Binding Parameters. Florescence quenching data was analyzed to obtain various binding parameters for the inter- 2.1. Fluorescence Quenching. BSA has two tryptophan action of Evo and BSA. e procedure of the �uorescence residues that possess intrinsic �uorescence: Trp-134 in the quenching was �rst assumed to be a dynamic quenching �rst domain and Trp-212 in the second domain. Tryptophan process. emission dominates BSA �uorescence spectra in the �V e dynamic quenching constant sv and the apparent region. [15, 16]. When other molecules interact with BSA, bimolecular quenching rate constant were calculated with tryptophan �uorescence may change depending on the the following Stern-Volmer equation [22]:𝐾𝐾 impact of such interaction on the protein conformation. 𝐾𝐾𝑞𝑞 e �uorescence intensity of BSA-Evo system was measured (1) with a pH of 7.34 and two different temperatures of 298 sv and 310 K. e effects of Evo on the �uorescence of BSA at 𝐹𝐹0 where and are= 1the + 𝐾𝐾relative𝑞𝑞𝜏𝜏0 [𝑄𝑄 �uorescence] = 1 + 𝐾𝐾 [𝑄𝑄 intensities] , in the temperatures of 298 K and 310 K are shown in Figure 2. e absence and presence𝐹𝐹 of quencher, [ ] is the concentration intensity of the characteristic broad emission band at 377 nm of quencher,0 is the Stern-Volmer dynamic quenching decreases markedly with the increasing concentration of 𝐹𝐹 𝐹𝐹 sv constant, is the bimolecular quenching𝑄𝑄 rate constant, and Evo, indicating that an interaction between Evo and BSA is the average bimolecular lifetime in the absence of has occurred, and the variation in intensity may result from 𝐾𝐾 quencher𝑘𝑘 evaluated𝑞𝑞 at about 5 ns. e plot of versus the changed protein conformation or direct quenching 0 𝜏𝜏[ ] gives a straight line, and sv is thus obtained from effect by Evo [17, 18]. However, the maximum emission the slope. Plots of versus [ ] are shown in0 Figure 4, wavelength of BSA barely changed during the interaction. 𝐹𝐹 /𝐹𝐹 and𝑄𝑄 the calculated sv and 𝐾𝐾are listed in Table 1. e As a result, we predict that polyamine binds mainly with the 0 values of obtained𝐹𝐹 are/𝐹𝐹 𝑄𝑄 L mol s at 298 K and two �uorophores Trp-212 buried inside and Trp-134 located 𝑞𝑞 on the surface of BSA, indicating that Trp-212 located within L mol s𝐾𝐾 at 310 K,𝑘𝑘 which12 are−1 much−1 larger than 𝑞𝑞 a hydrophobic binding pocket of the protein is not exposed the maximum𝑘𝑘12 scattering−1 −1 4.86 collisional × 10 quenching constant of to any change in polarity [19]. Similarly, Trp 134 located 2.41various × 10 quenchers of L mol s , indicating that the in this subdomain region would probably place it in the probable quenching mechanism10 of the−1 intrinsic−1 �uorescence hydrophobic packing interaction between helices and close of BSA was not initiated2.0×10 by a dynamic process but a static to the “distal” opening of the IB site. is suggests that the quenching procedure [23]. evodiamine is binding in the “proximal” IB side, and the e apparent association constant and the number of same binding mode of large heterocyclic molecules similar binding site were calculated using [22] to camptothecin with HSA is described in the published 𝐾𝐾 literature [20]. 𝑛𝑛 (2) Synchronous �uorescence spectra show Trp residues of BSA only at the wavelength interval of 60 nm and Tyr 󶀡󶀡𝐹𝐹0 − 𝐹𝐹󶀱󶀱 where andlogare󶁦󶁦 the apparent󶁶󶁶 = log association 𝐾𝐾 𝐾𝐾𝐾𝐾𝐾 constant[𝑄𝑄] , and the residues of BSA only at of 15 nm. Synchronous �uores- 𝐹𝐹 cence spectra of BSA-Evo are shown inΔ𝜆𝜆 Figure 3. It is apparent number of binding sites. Plots of log [ ] versus log that the intensity of TrpΔ𝜆𝜆 or Tyr residues decreases in the [ ] are𝐾𝐾 shown𝑛𝑛 in Figure 5. and were thus obtained from presence of Evo. However, the emission peak position of Trp the intercept on the -axis and the slope,(𝐹𝐹0 − respectively. 𝐹𝐹퐹�𝐹𝐹 e or Tyr residues does not show signi�cant shi�, indicating that calculated𝑄𝑄 and for Evo𝐾𝐾 at two𝑛𝑛 temperatures are listed in the polarity around Trp or Tyr residues is unchangeable [21]. Table 2. e medium𝑦𝑦 association constants of ( 𝐾𝐾 𝑛𝑛 6 𝐾𝐾 1.61 × 10 Journal of Chemistry 3

600 500

500 400

400 300 300 200 200 100 Fluorescence intensity Fluorescence 100 intensity Fluorescence 0 0

350 400 450 500 350 400 450 500 Wavelength (nm) Wavelength (nm)

(a) (b)

F 2: Emission spectra of BSA with various amounts of Evo at 298 K (a) and 310 K (b), BSA ( M): 10, Evo ( M): 4, 8, 12, 16, 20, 24, 28, 32, and 36. 𝐶𝐶 𝜇𝜇 𝐶𝐶 𝜇𝜇 60 600

480 45

360 30 240

15

Fluorescence intensity Fluorescence 120 Fluorescence intensity Fluorescence

0 0

270 285 300 315 330 290 295 300 305 310 Wavelength (nm) Wavelength (nm)

(a) (b)

F 3: Synchronous �uorescence spectra of BSA with various amounts of Evo, (a) nm, (b) nm. BSA ( M): 10 and Evo ( M): 4, 8, 12, 16, 20, 24, and 28. K. Δ𝜆𝜆 휆휆휆 Δ𝜆𝜆 휆휆휆 𝐶𝐶 𝜇𝜇 𝐶𝐶 𝜇𝜇 𝑇𝑇 � 푇푇푇 at 298 K and at 310 K) suggest that the affinity of T 1: Stern-Volmer quenching constants for BSA interaction Evo for BSA is just at5 a moderate level compared with the with Evo, is the correlation coefficient. reported binding6.78 constants× 10 of 10 –10 , in which Sandip et al. sv 𝑅𝑅 (K) 4 have reported that serum albumin4 has8 a limited number of ( 10 L/mol) ( 1012 L mol 1 s 1) binding sites for endogenous and exogenous ligands that are 𝑞𝑞 𝐾𝐾 𝐾𝐾 − − 𝑇𝑇298 4.96 4.86 0.9874𝑅𝑅 typically bound reversibly [24]. For example, baicalein which Evo-BSA × × is bound to BSA in the presence of Fe has a lower affinity 310 2.41 2.41 0.9806 ( L/mol) [15], while epicatechin3+ gallate binds to BSA in a higher4 binding constant of L/mol [25]. e T 2: Binding parameters for BSA interaction with Evo, is the values4.85 × of10 are 1.31 at 298 K and 1.33 at 3107 K, suggesting correlation coefficient. 𝑅𝑅 that 1-2 Evo molecules bound with6. BSA6 × 10 per protein. It is (K) (L/mol) indicated that this number ( ) of binding site does not depend 𝑛𝑛 298 1.31 0.9948 on the association constant ( ). Evo-BSA 𝑇𝑇310 𝐾𝐾 6 1.33𝑛𝑛 0.9938 𝑅𝑅 𝑛𝑛 1.61 × 105 2.3. Fluorescence Resonance𝐾𝐾 Energy Transfer. e rate of 6.78 × 10 energy transfer depends on the extent of the overlapping of BSA, the donor emission spectrum with the acceptor and acceptor transition dipoles, and the distance between absorption spectrum, the relative orientation of the donor these molecules [26]. 4 Journal of Chemistry

2.8 0.8 600

2.4 500 (1) 0.6

400 2 0.4 300

/F 0 F 200 0.2 1.6 Absorbance (2)

100 intensity Fluorescence 0 1.2 0

340 360 380 400 420 440 0 1 2 3 4 Wavelength (nm) [Q]×105 (mol/L) F 6: �verlap between the �uorescence emission spectrum of 298 K BSA (1) and UV absorption spectrum of Evo (2), BSA Evo 310 K M, K. 𝐶𝐶 = 𝐶𝐶 = F 4: Sterne-Volmer plot of the �uorescence quenching of BSA 10 𝜇𝜇 𝑇𝑇 𝑇�푇� with various amounts of Evo at 298 K (•) and 310 K ( ). T 3: e calculated values of , , , and of BSA with Evo, is the correlation coefficient. 0 3 1 𝐽𝐽 𝑟𝑟 𝑅𝑅 𝐸𝐸 𝑅𝑅 ★ (cm L mol ) ( ) (nm) (nm) 0.3 Evo-BSA − 0.41 1.67 1.57 0 𝐽𝐽 −14 𝐸𝐸 % 𝑅𝑅 𝑟𝑟 8.53 × 10 0 where the spatial orientation factor of the dipole ] , the refractive index of medium , and2 the ) /F F

- −0.3 �uorescence quantum yield of donor [27, 28].𝐾𝐾 can= 0 F 2/3be evaluated by the overlap of the UV absorption𝑁𝑁 𝑁𝑁𝑁 spectra of acceptor with the �uorescence emission𝜑𝜑 𝜑𝜑𝜑 spectra of donor.𝐽𝐽 is Log [( Log −0.6 given by the following equation: 𝐽𝐽 −0.9 (5) 4

−5 −4.8 −4.6 −4.4 ∑ 𝐹𝐹 (𝜆𝜆) 𝜀𝜀 (𝜆𝜆) 𝜆𝜆 Δ𝜆𝜆 e overlap between𝐽𝐽 � the BSA emission. spectra with the ∑ 𝐹𝐹 (𝜆𝜆) Δ𝜆𝜆 Log (Q) absorption spectra of Evo is shown in Figure 6, and the 298 K calculated values of , , , and are listed in Table 3. It is 310 K found that the value is 1.57 nm for Evo, which is less than the average distance of 2–80 nm between a donor and acceptor, F 5: Logarithmic plot of the �uorescence quenching of BSA 𝐽𝐽 𝑟𝑟 𝑅𝑅 𝐸𝐸 indicating that the energy transfer occurred between BSA and with various amounts of Evo at 298 K (•) and 310 K ( ). 𝑟𝑟 Evo with great possibility [8, 29]. ★ e energy transfer efficiency ( ) can be used to evaluate 2.4. ermodynamic Parameters. e thermodynamic the distance ( ) between the ligands (acceptor) and BSA parameters at different temperatures were analyzed to characterize the acting forces dominating the interaction. (donor) in protein by Förster’s theory𝐸𝐸 of dipole-dipole energy e enthalpy ( ), free energy ( ), and entropy ( ) transfer as follows:𝑟𝑟 changes are calculated according to the following Van’t Hoff equation: (3) Δ𝐻𝐻 Δ𝐺𝐺 Δ𝑆𝑆 6 𝐹𝐹 𝑅𝑅0 𝐸𝐸 퐸 = 6 6 , where and are the �uorescence0 0 intensity of BSA in 𝐹𝐹 󶀢󶀢𝑅𝑅 + 𝑟𝑟 󶀲󶀲 2 1 2 the presence and absence of the acceptor, is the distance 𝐾𝐾 󶀡󶀡1/𝑇𝑇 퐸 퐸/𝑇𝑇 󶀱󶀱 (6) ln = Δ𝐻𝐻 , between𝐹𝐹 the acceptor𝐹𝐹0 and donor, is the critical distance 𝐾𝐾1 𝑅𝑅 for 50 energy transfer which can be calculated𝑟𝑟 using the following: 𝑅𝑅0 Δ𝐺𝐺 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 % where is the associationΔ𝐺𝐺 constant𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 at temperature and is (4) the gas constant. e results are summarized in Table 4. Four 6 −25 2 −4 𝐾𝐾 𝑇𝑇 𝑅𝑅 𝑅𝑅0 = 8.8 × 10 𝐾𝐾 𝑁𝑁 Φ𝐽𝐽� Journal of Chemistry 5

T 4: e temperature-dependent thermodynamic parameters 4.2. Measurements. UV-Vis absorption spectra were per- for the Evo-BSA system. formed on a Varian Cary100 UV-Visible spectrophotome-

1 1 ter. Fluorescence measurements were performed on a T (K) (kJ/mol) (kJ/mol) (J mol K ) FluoroMax-4 spectrophotometer. 298 2.32 60.13 209.59− − Evo-BSA Δ𝐺𝐺 Δ𝐻𝐻 Δ𝑆𝑆 ⋅ 310 2.42 60.13 201.78 4.2.1. UV-Vis Absorption Spectra. UV-Vis absorption spectra − were performed on a Varian Cary100 UV-Visible spectropho- − tometer from 190 nm to 500 nm. types of acting forces including hydrogen bond, van der Waals 4.2.2. Fluorescence Emission Titration. Fluorescence emission force, electrostatic force, and hydrophobic interaction force spectra were then measured at 298 K or 310 K with a may be involved in the interaction between a small molecule pH of 7.34 and were recorded in a wavelength range of and a protein [30]. e positive value of and for Evo 290–500 nm. Samples were excited at 280 nm. Fluorescence suggests that hydrophobic and electrostatic interactions may emission spectra of compounds were performed by using be involved in the association and contributeΔ𝐻𝐻 to theseΔ𝑆𝑆 changes a �xed concentration of BSA (10 M) and increasing the [31]. e negative value of indicates that the interaction concentration of Evo (4, 8, 12, 16, 20, 24, 28, 32, and 36 M). of Evo with BSA is a spontaneous process. 𝜇𝜇 Δ𝐺𝐺 4.2.3. e Synchronous Fluorescence Spectra. Synchronous𝜇𝜇 3. Conclusions �uorescence spectra were obtained by simultaneously scan- ning the excitation and emission monochromators. e In the paper, the �uorescence quenching mechanism and wavelength interval ( ) at 15 and 60 nm was recorded over binding mode of evodiamine with bovine serum albumins a wavelength range of 240–360 nm by using a �xed concen- were investigated. e experimental results showed that tration of BSA (10 M)Δ𝜆𝜆 and increasing the concentration of Evo binds with BSA via a static quenching procedure with Evo (4, 8, 12, 16, 20, 24, and 28 M) at 298 K. association constants of L/mol at 298 K and 𝜇𝜇 L/mol at 310 K. e number6 of bound Evo molecules Acknowledgments 𝜇𝜇 per5 protein was 1.31𝐾𝐾 at 2981.61×10 K and 1.33 at 310 K. e results6.78× 10suggest that the Evo seems to react with BSA chie�y through e authors are grateful for the �nancial support from hydrophobic and electrostatic interactions, and it does not the National Natural Science Foundation of China (no. alter the -helical nature of BAS. e distance ( ) between 21261025, 81060360), the Natural Science Foundation of the ligands (acceptor) and BSA (donor) in protein is 1.57 nm Guangxi Province (no. 013064), the Key Laboratory for for Evo, which𝛼𝛼 is less than the average distance𝑟𝑟 of 2–8 nm the Chemistry and Molecular Engineering of Medicinal between a donor and acceptor, indicating that the energy Resources (Guangxi Normal University), and the Ministry transfer occurred between BSA and Evo with great possibility. of Education of China (no. CMEMR2011-09), as well as e positive value of and for Evo suggests that the talent training scheme of Guangxi Institutions of Higher hydrophobic and electrostatic interactions may be involved in Learning (2011-13-2). the association and contributeΔ𝐻𝐻 to theseΔ𝑆𝑆 changes. e negative value of indicates that the interaction of Evo with BSA is References a spontaneous process. Δ𝐺𝐺 [1] M. Ogasawara, T. Matsubara, and H. Suzuki, “Inhibitory effects of evodiamine on in vitro invasion and experimental 4. Experimental lung metastasis of murine colon cancer cells,” Biological and Pharmaceutical Bulletin, vol. 24, no. 8, pp. 917–920, 2001. 4.1. Materials. Evodiamine (Evo) was isolated from the [2] J. Yamahara, T. Yamada, T. Kitani, Y. Naitoh, and H. Fujimura, unripe fruit of Evodia rutaecarpa (Juss.) 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