Monatsh Chem (2013) 144:739–746 DOI 10.1007/s00706-012-0852-z

ORIGINAL PAPER

Interaction between diethylstilbestrol and bovine serum albumin Hanwen Sun • Yujie Wu • Xianghua Xia Xuyang Liu • Zhihong Shi

•

Received: 14 May 2012 / Accepted: 23 August 2012 / Published online: 21 September 2012 Ó Springer-Verlag 2012

Abstract The interaction between diethylstilbestrol (DES) and bovine serum albumin (BSA) was studied by fluorescence spectroscopy combined with the UV-Vis spectrophotometric technique under simulative physiological conditions. The influence of Cd and/or Se ions on the interaction between DES and BSA was also investigated. The fluorescence quenching rate constants, binding constants, and thermodynamic parameters for the BSA–DES system were determined at different temperatures. The distance between BSA and DES was estimated to be 3.65 nm based on the Fo¨rster resonance energy transfer theory. The fluorescence quenching of BSA by addition of DES is due to static quenching and energy transfer. The negative value of enthalpy change and entropy change indicated that both hydrogen bonding and van der Waals forces played major roles in the binding of DES to BSA. The tryptophan residue of BSA molecules mainly participated in the binding reaction to DES. Competitive experiments with warfarin, ibuprofen, and digoxin as specific probes suggested that the primary binding site for DES was located at site III in the sub-domain IIIA of BSA. In addition, the interaction between DES and BSA in the presence of Cd(II) decreased by four orders of magnitude. Keywords Fluorescence spectroscopy  Bovine serum albumin  Diethylstilbestrol  Cadmium  Selenium  Interaction

H. Sun (&)  Y. Wu  X. Xia  X. Liu  Z. Shi College of Chemical and Environmental Sciences, Hebei University, Key Laboratory of Analytical Science and Technology of Hebei Province, Baoding 071002, People’s Republic of China e-mail: [email protected]

Introduction Serum albumin is one of the most abundant proteins in the circulatory system of a wide variety of organisms and one of the most extensively studied proteins. The albumins make a significant contribution to colloid osmotic blood pressure and aid in the transport, distribution, and metabolism of many endogenous and exogenous ligands. Proteindrug binding greatly influences the absorption, distribution, metabolism, and excretion properties of typical drugs [1, 2]. Thus, it is important and necessary to study the interaction of drugs with serum albumins at the molecular level. Diethylstilbestrol (DES) is used as an adjunct palliative treatment in certain patients with breast and prostate cancer. Its pharmacological, toxicological, and carcinogenic properties were reviewed [3]. DES was etiologically linked to clear cell adenocarcinoma of the vagina. Hammes and Laitman [4] reviewed ongoing research and emerging information relevant to DES-related health risks, thereby enabling women’s health care providers to maintain an evidence-based practice for their DES-exposed patients. To date, spectroscopy, circular dichroism spectroscopy, and Fourier transformation infrared spectroscopy have been widely employed to study the interactions of small molecules and protein, and to investigate the conformational changes of protein under physiological conditions because of its accuracy, sensitivity, rapidity, and convenience [5–8]. It can reveal the accessibility of drugs to albumin’s fluorophores, which can help us to understand the binding mechanisms of albumin-drug and to provide information on the structural features for determining the therapeutic effectiveness of the drug. However, no reports have been made on the interactions of DES and protein. Human and bovine serum albumins exhibit similar binding chemistries because of the high percentage of

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sequence identities between the two proteins [9]. In this work, bovine serum albumin (BSA) was used because of its low cost and easy availability, and DES was used as a model drug. This study examined for the first time the interaction between BSA and DES under physiological conditions by fluorescence quenching in combination with the UV-Vis spectroscopic method.

Results and discussion Fluorescence quenching mechanism Fluorescence quenching refers to any process that decreases the fluorescence intensity of a sample. A variety of molecular interactions can result in fluorescence quenching of excited state fluorophores. These include molecular rearrangements, energy transfer, ground state complex formation, and collisional quenching. Figure 1 shows the fluorescence spectra of BSA in the absence and presence of DES. No fluorescence of DES was observed from threedimensional fluorescence spectra in a region of 280–800 nm. The fluorescence spectra of BSA show a broad band with a maximum at *340 nm. The fluorescence intensity of BSA was observed to decrease with increasing concentrations of DES. A maximum fluorescence emission underwent a spectral shift from 340 to 337 nm. It is suggested that an energy transfer between DES and BSA occurred. The quenching of fluorescence may be static or dynamic and can be recognized by temperature-dependence studies. The quenching rate constants are expected to decrease with

H. Sun et al.

increases in temperature for static quenching, while for dynamic quenching the reverse effect was observed. The fluorescence quenching data are analyzed by the SternVolmer equation [10]: F0 =F ¼ 1 þ Kq s0 ½Q ¼ 1 þ Ksv ½Q

ð1Þ

where F0 and F are the fluorescence intensity in the absence and presence of quencher, respectively. kq is the quenching rate constant, s0 is the fluorescence life time of biopolymer BSA (s0 = 10-8 s) [11], and Ksv and [Q] are the Stern-Volmer quenching constant and concentration of quencher, respectively. In this work, using 2.0 9 10-6 M BSA solutions with DES of 0, 0.149, 0.373, 0.745, 1.49, and 2.24 9 10-5 M, the Stern-Volmer equation of F0/F versus the concentration of DES was obtained at various temperatures. The estimated values of kinetic data along with the correlation coefficient are given in Table 1. The variation of F0/F against DES concentration fits in the equation of y = mx ? c with correlation coefficient (r) greater than 0.997. Kq was very much higher than the maximum dynamic quenching constant of various quenchers, 2.0 9 1010 M s-1 [12]. Based on the Kq and r values, it can be concluded that the quenching is not initiated by dynamic quenching but probably by static quenching. The shift in emission wavelength from 340 to 337 nm further indicates the formation of complex by binding of DES with BSA sites. Binding constant and binding site number The binding of DES with BSA to form a complex in the ground state is further understood on the basis of the available binding site number and binding constant of the complex formation process. For static quenching, the following equation was used to calculate the binding constant and binding sites [13, 14]: lg½ðF0  FÞ=F ¼ lg KA þ n lg½Q

ð2Þ

where KA and n are the binding constant and binding site number, respectively. The plots of lg[(F0 - F)/F] versus lg[Q] presented in Fig. 2 at different temperatures are linear. Figure 2 also shows the binding constant (KA) and the binding site number (n) calculated from the intercept and slope, along with regression coefficient (r). Table 1 Quenching reactive parameter of BSA–DES at different temperatures

Fig. 1 Quenching fluorescence spectra of BSA–DES. a–f BSA 2.0 9 10-6M, DES 0, 0.149, 0.373, 0.745, 1.49, 2.24 9 10-5 M, g DES 2.0 9 10-6 M

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T/K

Ksv/dm3 mol-1

Kq/dm3 mol-1 s-1

r

298

1.24 9 105

1.24 9 1013

0.9983

303

5

1.20 9 10

1.20 9 1013

0.9971

308

1.17 9 105

1.17 9 1013

0.9972

Interaction between diethylstilbestrol and bovine serum albumin

741

Fig. 2 Plot of lg[(F0 - F)/F] versus lg[Q]. BSA 2.0 9 10-6 mol dm-3 Fig. 3 Van’t Hoff plot for the interaction of DES with BSA at pH 7.40

Regression coefficient values nearly equal to one indicate the validity of Eq. (2). The table also shows that there was a stronger combination action between DES and BSA, and the binding constant changes very slightly with temperature. This observation suggests that the binding site does not change with temperature. Therefore, the present studies involve binding of a single molecule of DES with one molecule of BSA. Interaction forces between DES with BSA The interaction forces between drug and biomolecules include hydrogen bonds, van der Waals forces, and electrostatic and hydrophobic interactions [15]. The temperature dependence of the interaction of DES with BSA was investigated at 298, 303, and 308 K. The thermodynamic parameters can be evaluated from the van’t Hoff equation: ln K ¼ DH=RT þ DS=R

Table 2 Thermodynamic parameters of the interaction between DES and BSA at different temperatures T/K

KA/ dm3 mol-1

DH/ kJ mol-1

DS/ J mol-1 K-1

DG/ kJ mol-1

r

298

2.49 9 106

-71.54

-118.6

-36.20

0.9982

303

1.19 9 10

6

-35.60

308

9.41 9 105

-35.01

it. So van der Waals interaction and hydrogen bonding play major roles in the binding process [16]. Binding site of DES on BSA The binding site of DES on BSA was examined. Figure 4 shows that the fluorescence quenching of BSA by DES was observed under excitation at both 280 and 295 nm.

ð3Þ

where K is the binding constant at corresponding temperature T, and R is the gas constant. The enthalpy change (DH) and entropy change (DS) can be obtained from the slope and the ordinates at the origin of the van’t Hoff plot, respectively (Fig. 3). The free energy change DG is determined from the following relationship. DG ¼ DH  TDS

ð4Þ

The values of DG, DS, and DH are calculated and summarized in Table 2. The negative values of free energy (DG) support the assertion that the binding process is spontaneous. The negative enthalpy (DH) and entropy (DS) values of the interaction of DES and BSA indicate that the binding is mainly enthalpy-driven and the entropy is unfavorable for

Fig. 4 Fluorescence quenching curves of 2.0 9 10-6 M BSA in the presence of DES at 298 K and kem = 340 nm

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The fluorescence of BSA is mostly from tyrosine (Tyr) and tryptophan (Trp) residues at the 280-nm excitation wavelength, and the fluorescence of BSA is only from the tryptophan residue (Trp) at the 295-nm excitation wavelength. The two curves nearly overlapped, suggesting that the tryptophan residue mainly participates in the binding reaction between DES and BSA. On the basis of the probe-displacement method, there are at least three relatively highly specific drug-binding sites on the BSA molecules. These sites, commonly called the warfarin-, ibuprofen-, and digoxin-binding sites, are also denoted as site I, site II, and site III, respectively [17, 18]. To further determine the binding site of DES, competitive experiments were carried out at a temperature of 298 K using warfarin, ibuprofen, and digoxin as a site I-, site II-, and site III-specific probe, respectively. The concentration ratio of BSA and probe was 1:1 (2 9 10-6 M:2 9 10-6 M), concentration of DES was in the range of 7.453 9 10-8 to 2.236 9 10-7 M. Plots of lg[(F0 - F)/F] versus lg[CDES] in the absence and presence of site specific probe were prepared. The values of binding constant (KA) and the binding site number (n) were calculated from the intercept and slope based on Eq. (2). The binding constant and the binding site number were obtained. The data in Table 3 show that the binding constants for the DES–BSA system and binding site number were 2.49 9 106 dm3 mol-1 and 1.34, while in the presence of site-specific probe, the binding constants and binding site number decreased obviously. In the digoxin probe case, the binding constants decreased to 2.31 9 104 dm3 mol-1, two orders of magnitude lower than that for the DES–BSA system without any specific probe, and the binding site number decreased from 1.34 to 0.83. The competition of digoxin with DES at a same site was shown. The competitive experiments suggested that the primary binding site of DES on BSA was located at site III in sub-domain IIIA of BSA. Energy transfer from BSA to DES Fluorescence resonance energy transfer is an important technique for investigating a variety of biological phenomena, including energy transfer processes [19]. Here the

donor and acceptor are BSA and DES, respectively. The fluorescence emission of BSA–DES solution at an excitation wavelength of 280 nm is from BSA only since DES is a non-fluorescent drug molecule. However, at this wavelength DES has weak absorption. It was observed that there is spectral overlap between fluorescence emission of BSA and absorption spectra of DES in the wavelength range of 260–500 nm, as shown in Fig. 5. It suggested the possibility of fluorescence resonance energy transfer from BSA to DES molecules in solution. The region of integral overlap is used to calculate the critical energy transfer distance (R0) between BSA (donor) and DES (acceptor) according to Foster’s non-radioactive energy transfer theory using Fo¨rster’s equation [13, 20]. Based on this theory, the efficiency (E) of energy transfer between the donor (BSA) and acceptor (DES) can be calculated by Eq. (5): E ¼ R60 =ðR60 þ r 6 Þ

ð5Þ

where r is the binding distance between the donor and acceptor, and R0 is the critical binding distance when the efficiency (E) of the energy transfer is 50 %, which can be calculated by Eq. (6): R60 ¼ 8:8  1025 k2 n4 UD J

ð6Þ

where k2 is the spatial orientation factor of the dipole, n the refractive index of medium, UD the quantum yield of the donor in the absence of an acceptor, and J the overlap integral of the emission spectrum of the donor and the absorption spectrum of the acceptor. J can be calculated by Eq. (7): .X X J¼ FðkÞeðkÞk4 Dk FðkÞDk ð7Þ where F(k) is the fluorescence intensity of the fluorescent donor of wavelength k, and e(k) is the molar absorption coefficient of the acceptor at wavelength k. In the present case, k2, n, and UD are 2/3, 1.336, and 0.15, respectively [21]. The efficiency (E) of energy transfer can be determined by Eq. (8): E ¼ 1  F=F0

ð8Þ

where F0 and F are the fluorescence intensities of BSA solutions in the absence and presence of DES, respectively.

Table 3 Linear equation, binding constant KA, and binding site number n between DES and BSA in the presence of site-specific probe System

Linear equation

r

KA/dm3 mol-1

n

DES–BSA

lg[(F0 - F)/F] = 6.40 ? 1.34 lg[Q]

0.9996

2.49 9 106

1.34

DES–BSA–warfarin

lg[(F0 - F)/F] = 5.70 ? 1.15 lg[Q]

0.9980

5.02 9 105

1.15

DES–BSA–ibuprofen

lg[(F0 - F)/F] = 5.74 ? 1.14 lg[Q]

0.9984

5.56 9 105

1.14

DES–BSA–digoxin

lg[(F0 - F)/F] = 4.36 ? 0.83 lg[Q]

0.9981

2.31 9 104

0.83

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Interaction between diethylstilbestrol and bovine serum albumin

743

residue and tryptophan residue did not change during the binding process [26]. Effect of Se and Cd ions on binding constant

Fig. 5 Overlap spectra of DES absorption with BSA fluorescence. BSA 1 9 10-5 M, DES 1 9 10-5 M

From the overlapping, we found R0 = 2.93 nm from Eq. (6) using k2 = 2/3, n = 1.336, and UD = 0.118 (tryptophan residue) for the aqueous solution of BSA. J could be calculated from Eq. (7), and the corresponding result was 2.928 9 10-14 cm3 mol-1. E calculated from Eq. (8) was 0.2109. At the same time, the binding distance (r) between BSA and DES is obtained by Eq. (5), and the obtained result is 3.65 nm. Apparently, it is \7.0 nm. This result indicates that the non-radiative energy transfer from BSA to DES occurs with high possibility [22, 23]. It also suggested that the bindings of DES to BSA molecules were formed through energy transfer, which quenched the fluorescence of BSA molecules, indicating the presence of a static quenching interaction between BSA and DES [24].

Metal ions, especially those of bivalent type, are vital to the human body and play an essentially structural role in many proteins based on coordinate bonds [12, 27]. A lower concentration of Cu(II) had good effects in improving the efficacy of colistin sulfate on BSA [28]. Cd (II) could interact with both Trp 214 in sub-domain IIA and Trp 135 in sub-domain IB of BSA [29, 30]. To investigate the effect of coexistent ions, the binding constants were investigated in the presence of Cd (II) or/and Se(IV). The plots of lg [(F0 - F)/F] versus lg [Q] presented in Figs. 7 and 8 are linear. The values of the modified binding constants K0 of BSA–DES are listed in Table 4. Table 4 shows that the binding constant of the DES– BSA complex decreased in the presence of the ions. In the presence of 1 9 10-4 M Se(IV) and Cd(II) ions alone, the binding constant decreased from 5.16 9 106 to 1.20 9 106 and 3.09 9 102, respectively. The decrease in the binding constant reveals that the interaction between BSA and DES decreases since there is a competition of the ions with DES in the DES–BSA binding process. In the presence of 1 9 10-4 M Cd(II) alone and Cd(II) plus Se(IV) ions with each 1 9 10-4 M, the fluorescence peak of the DES–BSA system was removed from 340 to 328 nm. The binding constant of DES–BSA decreased by four orders of magnitude, and the binding site number decreased by 50 %. It is due to some microenvironmental and conformational changes of BSA molecules in the presence of Cd(II). This may lead to the need for more doses of DES to achieve the desired therapeutic effect.

Effect of DES on the conformation of BSA Synchronous fluorescence is a kind of simple and sensitive method to measure the fluorescence quenching. It can provide information of the polarity change around the chromophore microenvironment. Dk, representing the difference between excitation and emission wavelengths, is an important operating parameter. When Dk is 15 nm, synchronous fluorescence is characteristic of the tyrosine residue, while when Dk is 60 nm, it provides the characteristic information of tryptophan residues [25]. The synchronous fluorescence spectra of the tyrosine residue and tryptophan residues in BSA with the addition of DES were observed, as shown in Fig. 6. The fluorescence intensity decreased regularly along with the addition of DES, and the maximum emission wavelength did not obviously change. It is indicated that the presence of DES did not change the conformation of BSA and that the microenvironment around the tyrosine

Conclusion The results showed that DES can strongly bind to BSA by hydrogen bonding and van der Waals forces. The primary binding site for DES was located at site III in sub-domain IIIA of BSA. The interaction between DES and BSA in the presence of Cd(II) decreased markedly. The results are of great importance in pharmacy, pharmacology, and biochemistry and are expected to provide important insights into the interactions of the physiologically important protein BSA with DES.

Materials and methods Commercially available BSA (purity [98 %) was purchased from Sigma Chemical Co. BSA stock solution

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Fig. 6 Synchronous fluorescence of the DES–BSA system at Dk = 15 nm (a) and Dk = 60 nm (b). BSA 2.0 9 10-6 M, DES, a–f 0, 0.149, 0.373, 0.745, 1.49, 2.24 9 10-5 M

Fig. 7 Plot of lg[(F0 - F)/F] versus lg CDES in the presence of Se (a) and Cd (b) at 298 K. Se or Cd: a 1 9 10-5 M, b 1 9 10-4 M, BSA 2 9 10-6 M

Table 4 Binding constant KA and binding site number n between DES and BSA at 298 K in the presence of different concentrations of Se and Cd ions Ions

C/M

–

–

n

0.9996

2.49 9 106

1.34

Se

1 9 10

0.9985

2.19 9 106

1.26

Cd

1 9 10-4 1 9 10-5

0.9998 0.9995

1.20 9 106 2.14 9 106

1.20 1.29

1 9 10-4

0.9983

3.09 9 102

0.51

0.9986

2.24 9 106

1.30

0.9980

8.13 9 102

0.60

1 9 10

-5 a

1 9 10-4 a

123

KA/dm3 mol-1

-5

Se ? Cd

Fig. 8 Plot of lg[(F0 - F)/F] versus lg CDES in the presence of Se plus Cd at 298 K. Se ? Cd: a each 1 9 10-5 M, b each 1 9 10-4 M, BSA 1 9 10-5 M

r

a

For each species

(1.0 9 10-4 M) was prepared by dissolving an appropriate amount of BSA with Tris-HCl (pH 7.4) buffer solution and kept in the dark at 4 °C. All BSA working solutions were prepared after dilution with water. A stock DES solution (1,000 lg cm-3) was prepared by directly dissolving an

Interaction between diethylstilbestrol and bovine serum albumin

appropriate amount of DES (purity [99.9 %) in diluted alcohol and diluting with diluted alcohol to 100.0 cm3. This solution was diluted to 1.0 9 10-5 M with water as a working solution. NaCl (analytical grade 0.5 M) solution was used to maintain the ion strength at 0.1 M. The buffer (pH 7.40) consists of Tris (0.1 M) and HCl (0.1 M). Warfarin, ibuprofen, and digoxin were purchased from the Control of Pharmaceutical and Biological Products (Beijing, China). Cd and Se stock solutions (1.0 9 10-3 M) were prepared with CdSO4 and Na2SeO3, respectively, and kept in the dark at 0–4 °C. All chemicals were of analytical reagent grade or better. All water used was doubledistilled. All fluorescence measurements were made with a F-7000 Fluorescence spectrophotometer (Hitachi, Japan) equipped with a 1-cm quartz cell and thermostat bath. The spectrum data points were collected from 300 to 400 nm. The widths of the excitation and the emission slit were both set at 5 nm. Fluorescence measurement was carried out at different temperatures. The absorption spectra were performed on a Shimadzu UV-1700 spectrophotometer using a 1-cm quartz cell in the wavelength range of 200–400 nm. All pH measurements were made with a pHS-3C pH meter (Shanghai, China). Determination of fluorescence intensity Five 10-cm3 clean and dried test tubes were taken, and 1 cm3 of 2 9 10-5 M BSA, 2 cm3 Tris-HCl buffer (pH 7.4), and different volumes (1–3 cm3) of DES solution of 7.453 9 10-5 M were taken in each of them, and diluted to the mark with water. A sixth test tube containing only BSA solution at pH 7.4 was marked as ‘‘control.’’ After mixing the solutions, these were allowed to stand for 10 min for maximum binding of DES to BSA. The fluorescence intensity (F0) in the absence of quencher DES and the fluorescence intensity (F) in the presence of quencher DES were measured at an excitation wavelength of 278 nm and temperature of 298, 303, and 308 K for estimating the interaction between DES and BSA. Determination of the fluorescence quenching constants, binding constant, and binding site number The Stern-Volmer quenching constants were determined based on the Stern-Volmer equation [10]. The binding constant and binding site number are determined according the equation in references [13, 14]. The binding constants of DES–BSA in the presence of Se(IV) and Cd(II) ions were determined according to the same method.

745

Determination of thermodynamic parameters Using the obtained binding constants at different temperatures, thermodynamic parameters and the nature of the binding forces between DES and BSA were estimated based on the van’t Hoff equation. Replacement experiments To determine the binding site of DES, warfarin sodium, ibuprofen, and digoxin were used as site I-, site II-, and site III-specific probes, respectively. BSA solution (1 cm3, 2 9 10-5 M), 1 cm3, 2 9 10-5 M probe solution, 2.0 cm3 of 0.5 M NaCl solution, and 2.0 cm3 Tris-HCl buffer solution (pH 7.40) were taken in each of the five 10-cm3 cleaned and dried test tubes, and the final ratio of BSA and probe was 1:1 (2 9 10-6 M:2 9 10-6 M) in each of these five test tubes. The sixth test tube containing only BSA solution was marked as ‘‘control.’’ Different volumes (1–3 cm3) of DES solution (7.453 9 10-5 M) were added with increasing concentrations into the five test tubes containing a 1:1 mixture of the BSA probe. These mixtures were allowed to stand for 10 min to allow binding of the probe to its particular binding site. The binding constant KB and the binding site number n for the system in the presence of the site probe were determined according to the equation in references [13, 14]. Determination of energy transfer efficiency and critical energy transfer distance The absorption spectra of DES and fluorescence spectra of BSA were determined by a UV-Vis spectrophotometer and fluorescence spectrophotometer, respectively. Spectral overlap of DES absorption with BSA fluorescence was observed. The region of integral overlap was used to calculate the efficiency (E) of the energy transfer and critical energy transfer distance (R0) between BSA and DES according to Foster’s non-radioactive energy transfer theory using Fo¨rster’s equation [13, 20]. Acknowledgments This work was supported by the Natural Science Found of Hebei Province (B2008000583) and the sustentation Plan of Science and Technology of Hebei Province (no. 10967126D).

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