Research Article Fluorescence Spectroscopy Study on the Interaction Between Evodiamine and Bovine Serum Albumin
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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, China 2 e Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry & Chemical Engineering, Guangxi 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 does not1.61 alter × the 10 -helical nature of BAS.6.78 × 10 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. 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]: impact of such interaction on the protein conformation. 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 0 where and are= 1the + relative0 [ �uorescence] = 1 + [ intensities] , in the temperatures of 298 K and 310 K are shown in Figure 2. e absence and presence 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 sv constant, is the bimolecular quenching 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 quencher evaluated at about 5 ns. e plot of versus the changed protein conformation or direct quenching 0 [ ] 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. / and the calculated sv and are listed in Table 1. e As a result, we predict that polyamine binds mainly with the 0 values of obtained are/ L mol s at 298 K and two �uorophores Trp-212 buried inside and Trp-134 located on the surface of BSA, indicating that Trp-212 located within L mol s at 310 K, which12 are−1 much−1 larger than a hydrophobic binding pocket of the protein is not exposed the maximum12 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 literature [20]. (2) Synchronous �uorescence spectra show Trp residues of BSA only at the wavelength interval of 60 nm and Tyr 0 − where andlogare the apparent = log association constant[] , and the residues of BSA only at of 15 nm. Synchronous �uores- cence spectra of BSA-Evo are shown inΔ Figure 3. It is apparent number of binding sites. Plots of log [ ] versus log that the intensity of TrpΔ or Tyr residues decreases in the [ ] are shown 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,(0 − respectively. 퐹/ e or Tyr residues does not show signi�cant shi�, indicating that calculated and for Evo at two temperatures are listed in the polarity around Trp or Tyr residues is unchangeable [21]. Table 2. e medium association constants of ( 6 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. 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. Δ 휆휆휆 Δ 휆휆휆 휆 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 at휆 a moderate level compared with the with Evo, is the correlation coefficient. reported binding휆.7푇 constants× 휆휆 of 10 –10 , in which Sandip et al. sv (K) 4 have reported that serum albumin4 has푇 a limited number of ( 10 L/mol) ( 1012 L mol 1 s 1) binding sites for endogenous and exogenous ligands that are − − 298 4.96 4.86 0.9874 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].