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

e interaction of evodiamine (Evo) with bovine serum albumins (BSAs) at different two temperatures (298 and 310K) was investigated bymeans of �uorescence spectroscopy.e experimental results showed that Evo bindswith BSA via a static quenching procedure with association constants KK of 1.61 × 10 L/mol at 298K and 6.78 × 10 L/mol at 310K. e number of bound Evo molecules per protein is 1.31 at 298K and 1.33 at 310K.e results suggested that Evo reacts with BSA chie�y through hydrophobic and electrostatic interactions, and it does not alter the αα-helical nature of BAS.


Introduction
Evodiamine (Figure 1), a quinolone alkaloid, is the major component isolated from the fruit of Evodia rutaecarpa, which is a traditional medicinal plant distributed in East Asia, especially in China, and has been used for a long time as a traditional Chinese medicine for the treatment of gastrointestinal disorders, headache, and postpartum hemorrhage.In pharmacology studies, it has been reported that evodiamine (Evo) was found to have antinociceptive, anti-in�ammatory, antiobesity, vasodilatory, thermoregulatory, analgesic, cardiotonic, uterotonic, and antitumor activities [1][2][3].Studies concerning screening of alkaloids have shown that evodiamine exhibits the strongest cytotoxicity activity against human colon and hepatoblastoma cell lines and inhibitory activity on human colon carcinoma cell.Further studies demonstrate that evodiamine has antitumor potential by inhibiting proliferation, inducing apoptosis and reducing invasion and metastasis of a wide variety of tumor cells, including breast cancer cells, prostate cancer cells, leukemic T-lymphocyte cells, melanoma cells, cervical cancer cells, colon cancer cells, and lung cancer cells.More importantly, evodiamine not only sensitizes chemoresistant breast cancer cells to adriamycin, but also shows little toxicity against normal human peripheral blood cells [4].
Serum albumin (SA) is a multiple function protein and acts as the transporter and disposition of many endogenous and exogenous ligands, including fatty acids, amino acids, metals ions, and numerous pharmaceuticals by means of hydrogen bonding, hydrophobic, electrostatic, and metal interactions [4][5][6][7].e interaction intensity between antitumor drugs and SA may effect on their bioavailability and toxicity [8][9][10].In this regard, bovine serum albumin (BSA) has been studied extensively, partly because of its structural homology with human serum albumin (HSA).BSA is composed of three linearly arranged, structurally homologous subdomains.It has two tryptophan residues that possess intrinsic domains, and each domain in turn is the product of two �uorescence: Trp134, which is located on the surface of subdomain IB, and Trp212, locating within the hydrophobic binding pocket of subdomain IIA.e binding sites of BSA for endogenous and exogenous ligands may be in these domains.
Fluorescence quenching is considered as a method for measuring binding affinities.Fluorescence quenching is the decrease of the quantum yield of �uorescence from a �uorophore induced by a variety of molecular interactions with quencher molecule [11,12].It is, therefore, of interest to use quenching of the intrinsic tryptophan �uorescence of BSA

Results and Discussion
2.1.Fluorescence Quenching.BSA has two tryptophan residues that possess intrinsic �uorescence: Trp-134 in the �rst domain and Trp-212 in the second domain.Tryptophan emission dominates BSA �uorescence spectra in the �V region.[15,16].When other molecules interact with BSA, tryptophan �uorescence may change depending on the impact of such interaction on the protein conformation.e �uorescence intensity of BSA-Evo system was measured with a pH of 7.34 and two different temperatures of 298 and 310 K. e effects of Evo on the �uorescence of BSA at temperatures of 298 K and 310 K are shown in Figure 2. e intensity of the characteristic broad emission band at 377 nm decreases markedly with the increasing concentration of Evo, indicating that an interaction between Evo and BSA has occurred, and the variation in intensity may result from the changed protein conformation or direct quenching effect by Evo [17,18].However, the maximum emission wavelength of BSA barely changed during the interaction.As a result, we predict that polyamine binds mainly with the two �uorophores Trp-212 buried inside and Trp-134 located on the surface of BSA, indicating that Trp-212 located within a hydrophobic binding pocket of the protein is not exposed to any change in polarity [19].Similarly, Trp 134 located in this subdomain region would probably place it in the hydrophobic packing interaction between helices and close to the "distal" opening of the IB site.is suggests that the evodiamine is binding in the "proximal" IB side, and the same binding mode of large heterocyclic molecules similar to camptothecin with HSA is described in the published literature [20].
Synchronous �uorescence spectra show Trp residues of BSA only at the wavelength interval Δ of 60 nm and Tyr residues of BSA only at Δ of 15 nm.Synchronous �uorescence spectra of BSA-Evo are shown in Figure 3.It is apparent that the intensity of Trp or Tyr residues decreases in the presence of Evo.However, the emission peak position of Trp or Tyr residues does not show signi�cant shi�, indicating that the polarity around Trp or Tyr residues is unchangeable [21].So, Evo does not obviously affect the -helical conformation of BSA.

Binding Parameters.
Florescence quenching data was analyzed to obtain various binding parameters for the interaction of Evo and BSA.e procedure of the �uorescence quenching was �rst assumed to be a dynamic quenching process.
e dynamic quenching constant  sv and the apparent bimolecular quenching rate constant   were calculated with the following Stern-Volmer equation [22]: where  0 and  are the relative �uorescence intensities in the absence and presence of quencher, [] is the concentration of quencher,  sv is the Stern-Volmer dynamic quenching constant,   is the bimolecular quenching rate constant, and  0 is the average bimolecular lifetime in the absence of quencher evaluated at about 5 ns.e plot of  0 / versus [] gives a straight line, and  sv is thus obtained from the slope.Plots of  0 / versus [] are shown in Figure 4, and the calculated  sv and   are listed in Table 1.e values of   obtained are 4.86 × 10 12 L mol −1 s −1 at 298 K and 2.41 × 10 12 L mol −1 s −1 at 310 K, which are much larger than the maximum scattering collisional quenching constant of various quenchers of 2.0×10 10 L mol −1 s −1 , indicating that the probable quenching mechanism of the intrinsic �uorescence of BSA was not initiated by a dynamic process but a static quenching procedure [23].e apparent association constant  and the number of binding site  were calculated using [22] where  and  are the apparent association constant and the number of binding sites.Plots of log [( 0 − /] versus log [] are shown in Figure 5.  and  were thus obtained from the intercept on the -axis and the slope, respectively.e calculated  and  for Evo at two temperatures are listed in  e energy transfer efficiency () can be used to evaluate the distance () between the ligands (acceptor) and BSA (donor) in protein by Förster's theory of dipole-dipole energy transfer as follows: where  and  0 are the �uorescence intensity of BSA in the presence and absence of the acceptor,  is the distance between the acceptor and donor,  0 is the critical distance for 50% energy transfer which can be calculated using the following: where the spatial orientation factor of the dipole  2  2/3, the refractive index of medium   .36, and the �uorescence quantum yield of donor   0.5 [27,28]. can be evaluated by the overlap of the UV absorption spectra of acceptor with the �uorescence emission spectra of donor. is given by the following equation: e overlap between the BSA emission spectra with the absorption spectra of Evo is shown in Figure 6, and the calculated values of , ,  0 , and  are listed in Table 3.It is found that the  value is 1.57nm for Evo, which is less than the average distance of 2-8 nm between a donor and acceptor, indicating that the energy transfer occurred between BSA and Evo with great possibility [8,29].

ermodynamic Parameters.
e thermodynamic parameters at different temperatures were analyzed to characterize the acting forces dominating the interaction.e enthalpy (Δ), free energy (Δ), and entropy (Δ) changes are calculated according to the following Van where  is the association constant at temperature  and  is the gas constant.e results are summarized in types of acting forces including hydrogen bond, van der Waals force, electrostatic force, and hydrophobic interaction force may be involved in the interaction between a small molecule and a protein [30].e positive value of Δ and Δ for Evo suggests that hydrophobic and electrostatic interactions may be involved in the association and contribute to these changes [31].e negative value of Δ indicates that the interaction of Evo with BSA is a spontaneous process.

Conclusions
In the paper, the �uorescence quenching mechanism and binding mode of evodiamine with bovine serum albumins were investigated.e experimental results showed that Evo binds with BSA via a static quenching procedure with association constants  of 1.61×10 6 L/mol at 298 K and 6.78× 10 5 L/mol at 310 K. e number of bound Evo molecules per protein was 1.31 at 298 K and 1.33 at 310 K. e results suggest that the Evo seems to react with BSA chie�y through hydrophobic and electrostatic interactions, and it does not alter the -helical nature of BAS.e distance () between the ligands (acceptor) and BSA (donor) in protein is 1.57nm for Evo, which is less than the average distance of 2-8 nm between a donor and acceptor, indicating that the energy transfer occurred between BSA and Evo with great possibility.e positive value of Δ and Δ for Evo suggests that hydrophobic and electrostatic interactions may be involved in the association and contribute to these changes.e negative value of Δ indicates that the interaction of Evo with BSA is a spontaneous process.

F 5 :
Logarithmic plot of the �uorescence quenching of BSA with various amounts of Evo at 298 K (•) and 310 K (★).

Table 2 .
e medium association constants of  (1.61 × 10 6 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 36.298 K and .7 ×   at 310 K) suggest that the affinity of Evo for BSA is just at a moderate level compared with the reported binding constants of 10 4 -10  , in which Sandip et al.
F 3: Synchronous �uorescence spectra of BSA with various amounts of Evo, (a) Δ   nm, (b) Δ   nm. BSA (M): 10 and  Evo (M): 4, 8, 12, 16, 20, 24, and 28.   K. at 2.3.Fluorescence Resonance Energy Transfer.e rate of energy transfer depends on the extent of the overlapping of BSA, the donor emission spectrum with the acceptor absorption spectrum, the relative orientation of the donor T 1: Stern-Volmer quenching constants for BSA interaction with Evo,  is the correlation coefficient.
)  BSA   Evo  0 M,   28 K. T 3: e calculated values of , ,  0 , and  of BSA with Evo,  is the correlation coefficient.