Interaction of the docetaxel with human serum albumin using optical spectroscopy methods

ARTICLE IN PRESS Journal of Luminescence 129 (2009) 1196–1203

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Interaction of the docetaxel with human serum albumin using optical spectroscopy methods Hongxia Cheng 1, Hui Liu, Yuying Zhang, Guolin Zou  State Key Laboratory of Virology, College of Life Sciences, Center of Nanoscience and Nanotechnology, Wuhan University, Wuhan 430072, China

a r t i c l e in f o

a b s t r a c t

Article history: Received 9 January 2009 Received in revised form 26 April 2009 Accepted 27 May 2009 Available online 13 June 2009

Docetaxel is a semi-synthetic product derived from the needles of the European yew. It is an antineoplastic agent belonging to the taxoid family. The interaction between docetaxel and human serum albumin (HSA) has been investigated systematically by the fluorescence quenching technique, synchronous fluorescence spectroscopy, ultraviolet (UV)–vis absorption spectroscopy, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) under physiological conditions. Our fluorescence data showed that HSA had only one docetaxel binding site and the binding process was a static quenching procedure. According to the Van’t Hoff equation, the thermodynamic parameters standard enthalpy (DH0) and standard entropy (DS0) were calculated to be 41.07 KJ mol1 and 49.72 J mol1 K1. These results suggested that hydrogen bond was the predominant intermolecular force stabling the docetaxel–HSA complex. The data from the CD, FT-IR and UV–vis spectroscopy supported the change in the secondary structure of protein caused by the interaction of docetaxel with HSA. & 2009 Elsevier B.V. All rights reserved.

Keywords: Docetaxel Human serum albumin Fluorescence quenching Circular dichroism FT-IR Binding thermodynamics

1. Introduction Docetaxel ((2R, 3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester, 13-ester with 5b, 20-epoxy-1, 2a, 4, 7b, 10b and 13a-hexahydroxytax-11-en-9-one 4-acetate 2-benzoate, trihydrate ) (Fig. 1) is a new taxoid, structurally similar to paclitaxel, but more effective as an inhibitor of microtubule depolymerization [1]. It is clinically effective against advanced breast, ovarian and non-small cell lung cancer [2]. Some chemotherapeutic drugs, when using with docetaxel, show higher anticancer efficacy in patients with breast, pancreatic, gastric and urothelial carcinomas than they were used alone [3–6]. Docetaxel is shown to be greater than 98% plasma protein bound at 37 1C and pH 7.4 [7]. Human serum albumin (HSA) is the most abundant protein of blood plasma. It has an important role in maintaining the colloidal osmotic pressure in blood. It also transports and distributes exogenous and endogenous substances, such as nutrients, hormones, fatty acids and many diverse drugs [8,9]. HSA is a globular protein consisting of 585 amino acid residues and considered to have three specific binding sites (labeled as I–III) [10]. Crystal

structure analysis has revealed site I and II reside in subdomains IIA and IIIA of HSA, respectively [11]. The interaction between drugs and plasma protein is of great interest, as it influences their pharmacokinetic and pharmacodynamic properties. Study on the docetaxel–HSA interaction mechanisms can provide the structural features that determine the therapeutic effectiveness of drug. The molecular interactions are often monitored by spectroscopic techniques [12], because these methods are sensitive and relatively easy to use. Compared to spectroscopic techniques, conventional methods such as equilibrium dialysis and ultrafiltration suffer from lack of sensitivity, long analysis time and use of protein concentrations far in excess of the dissociation constant for the drug–protein complex studies [13–17]. In this study, the binding of docetaxel to HSA was systematically investigated by using fluorescence spectroscopy and ultraviolet (UV) absorption spectrum. Furthermore, the effects of docetaxel on conformational changes of HSA were researched by circular dichroism (CD) and Fourier transform infrared (FT-IR). The study described a possible mechanism that explained how docetaxel and HSA interact with each other in vivo. 2. Experimental

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E-mail addresses: [email protected] (H. Cheng), zouguolin@whu. edu.cn (G. Zou). 1 Tel.: +86 27 81695654; fax: +86 27 81695555. 0022-2313/$ - see front matter & 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2009.05.023

2.1. Materials HSA was purchased from Sigma (St Louis, MO, USA). Docetaxel was purchased from Shanghai Winherb Medical Science Co., Ltd.

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were also measured and subtracted from the spectra of the sample.

Fig. 1. Structure of docetaxel.

(China). The stock solution of docetaxel was prepared in anhydrous ethanol and kept in the dark at 277 K. The molar concentration was based on its molecular weight of 807.8. The experiment phosphate buffer solution (PBS) was consisted of 0.01 mol l1 NaH2PO4–Na2HPO4 and 0.10 mol l1 of NaCl. The pH value of solution was kept at 7.40. The HSA solution was prepared immediately in PBS before use and the molar concentration was prepared based on its molecular weight of 66,500Da. All reagents were analytical reagent grade. Water from a Milli Q system apparatus (Millipore, USA) was used throughout the experiment. 2.2. Apparatus UV–vis absorption spectra were recorded on Cary-100 UV–vis spectrophotometer (Varian, USA) with 1 cm quartz cell. Fluorescence spectroscopy measurements were performed on F-4500 fluorescence spectrophotometer (Hitachi, Japan) equipped with a thermostatically controlled cell holder and a 1.0 cm quartz cell. Excitation and emission slit widths were 5 and 10 nm, respectively. The scan speed was 240 nm min1. CD spectra were recorded on JASCO J-820 spectropolarimeter (Japan Spectroscopic, Japan), using 0.1 cm cell at 0.2 nm intervals, the slit width was 5.0 nm and scan speed was 60 nm min1. FT-IR measurements were recorded at room temperature on a Nicolet 5700 FT-IR spectrometer (USA) equipped with attenuated total reflection (ATR) accessory, deuterated triglycine sulphate (DTGS) detector and KBr beam splitter. All spectra were taken with a resolution of 4 cm1 and using 60 scans. 2.3. Procedures 2.3.1. Fluorescence quenching measurements The excitation wavelength of 295 nm was used, and the emission spectra were recorded from 305 to 400 nm at three temperatures (283,300 and 310 K). Titration quenching experiments were carried out as follows: A 3 ml protein sample (2.0  106 mol l1) was placed in a quartz cell and was titrated by successive additions of docetaxel (concentration from 1.0 to 7.0  106 mol l1). The overall dilution did not exceed 1.0%. All solution was mixed thoroughly and kept 10 min before measurements. 2.3.2. UV–vis measurements The UV–vis measurements of HSA in the presence or absence of docetaxel were made in the range of 190–300 nm at 298 K. Protein concentration was fixed at 2.0  106 mol l1, while the drug concentration was varied from 4  106to 28  106 mol l1. As a control, the UV–vis spectra of buffer titrated with docetaxel,

2.3.3. FT-IR measurements For the spectra processing procedures, the background (containing all system components, except protein) were collected at the same condition. The FT-IR spectrum of free HSA was acquired by subtracting the absorption of PBS from the spectrum of the protein solution, and the spectrum of HSA after binding with the drug was obtained by subtracting the spectrum of free docetaxel form with the same concentration. The subtraction criterion was that the original spectrum of protein solution between 2200 and 1800 cm1 was featureless [18]. Fourier self-deconvolution and secondary derivative were applied to the range of 1600–1700 cm1 to estimate the number, position and width of component bands. Based on these parameters, a curve-fitting process was carried out to get the best Gaussian-shaped curves that fit the original protein spectrum. After indentifying individual bands with its representative secondary structure, the percentages of each secondary structure of HSA were calculated by the relative area of their respective component bands. 2.3.4. CD measurements The CD measurements of HSA in the presence or absence of docetaxel were made in the range of 200–260 nm with three scans averaged for each CD spectrum. HSA concentration was fixed at 2.0  106 mol l1, while the molar ration of protein to drug concentration was 1:6 and 1:12. 2.3.5. 1-anilino-8-naphthalenesulfonate (ANS)-binding experiment The excitation wavelength of 380 nm was used, the fluorescence emission spectra of ANS were recorded from 400 to 600 nm in the absence and presence of HSA. The ANS concentration was fixed at 10  106 mol l1, while the HSA concentration was varied from 1 106 to 8  106 mol l1 .The fluorescence quenching spectra of ANS–HSA complex were recorded from 400 to 600 nm by successive additions of docetaxel. Docetaxel concentration was from 4  106 to 40  106 mol l1, while the molar ration of ANS to HSA was 2:1. 2.3.6. Energy transfer The UV–vis measurement of docetaxel was made in the range of 300–500 nm at 298 K. The fluorescence of HSA were recorded from 300–500 nm at an excite wavelength of 295 nm, while the molar ration of protein to drug concentration was 1:1.

3. Results and discussion 3.1. Fluorescence studies of docetaxel with HSA The fluorescence measurements can give some information on the binding of small molecule substances to protein. Fig. 2 shows the fluorescence spectra of HSA in presence of docetaxel at different concentrations. Docetaxel caused concentrationdependent quenching of the intrinsic fluorescence of HSA, and slight blue shift (from 339 to 337 nm) of the maximum emission wavelength of HSA, while excitation at 295 nm. These results indicate that there was the interaction between docetaxel and HSA. It also implies that the conformational change may be induced in HSA by docetaxel. The excitation wavelength of 295 nm was chosen to avoid the interference fluorescence of tyrosine and phenylalanine residues [19].

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3000

Table 1 Binding parameters of HSA–docetaxel interaction.

a

Fluorescence Intensity

2500

T (K)

KSV (  104 l mol1)

R

Kq (  1012 l mol1)

K (  104 l mol1)

n

283 300 310

4.094 3.027 2.935

0.9959 0.9935 0.9985

4.094 3.027 2.935

9.80 3.38 2.18

1.07 1.02 0.97

h 2000 1500 1000

283K

1.24 1.22

500

300K

1.20

310K

1.18 0 320

340 360 Wavelength (nm)

380

400

Fig. 2. Fluorescence spectra of HSA in the presence of docetaxel at 298 K. The concentration of docetaxel : (a) 0; (b) 1 106 mol l1; (c) 2  106 mol l1; (d) 3  106 mol l1; (e) 4  106 mol l1; (f) 5  106 mol l1; (g) 6  106 mol l1; and (h) 7  106 mol l1. The concentration of HSA is 2.0  106 mol l1, pH 7.4.

F0/F

1.16 300

1.14 1.12 1.10 1.08 1.06 1.04 1.02 1

3

4

5

6

[Q] (10-6mol L-1)

2.5

a

0.5

Fig. 4. Stern–Volmer curves for the binding of docetaxel with HSA at 283, 300 and 310 K. The concentration of HSA is 2  106 mol l1.

0.4 Absorbance

h

2.0 Absorbance

2

1.5

0.3 0.2

3.3. Fluorescence quenching mechanism of HSA induced by docetaxel

0.1 0.0

1.0 180

200

220

240

260

Wavelength (nm)

0.5

In order to speculate the fluorescence quenching mechanism, the fluorescence quenching data at different temperatures (283, 300 and 310 K) were analyzed by the Stern–Volmer equation [21]: F0 ¼ 1 þ KSV ½Q  F

0.0 180

200

220

240

260

Wavelength (nm) Fig. 3. UV–vis absorption spectra of HSA in presence of docetaxel at 298 K (inset for docetaxel). Concentration of docetaxel : (a) 0; (b) 4  106 mol l1; (c) 8  106 mol l1; (d) 12  106 mol l1; (e) 16  106 mol l1; (f) 20  106 mol l1; (g) 24  106 mol l1; and (h) 28  106 mol l1. The concentration of HSA is 2.0  106 mol l1, pH 7.4.

3.2. UV–vis absorption spectra UV–vis absorption measurement is a simple way to explore the structure change and complex formation. Fig. 3 shows the UV–vis absorption spectra of docetaxel and HSA in the absence and presence of docetaxel. HSA has a strong absorbance with peak at about 208 nm and it represents the characteristics of a-helix structure of protein [20].The absorbance of HSA decreased with the increasing concentration of docetaxel. Furthermore, a slight red shifts of maximum peak position (from 208 to 210 nm) could also been observed with the addition of docetaxel. These results indicate that interaction occurred between docetaxel and HSA. The evidences from fluorescence and UV spectra both suggest that there were a docetaxel–HSA complex formation and HSA microenvironment change.

ð1Þ

where F0 and F are the steady-state fluorescence intensities in the absence or presence of quencher (docetaxel), respectively. KSV is the Stern–Volmer quenching constant and [Q] is the concentration of quencher. The values of KSV at different temperatures are shown in Table 1. The linearity of the F0/F versus [Q] plots is shown in Fig. 4. It can be found from Table 1, the quenching constant KSV decreased with rising temperature which indicates that the probable quenching mechanism of HSA was a static quenching procedure and complex between docetaxel and HSA may be formed. The formation of complex is further confirmed from the values of quenching rate constant, Kq, which are evaluated using the equation Kq ¼

KSV

t0

ð2Þ

where t0 is the average lifetime of the protein without the quencher. The value of t0 of the biopolymer is 108 s1 and value of Kq is at the order of 1012 l mol1 s1 for HSA. The maximum scatter collision quenching constant Kq of various quenchers with the biopolymer is 2  1010 l mol1 s1 [22]. Obviously, the Kq value initiated by docetaxel was greater than that of the scattered procedure. This further suggests that the quenching mechanism was mainly arisen from the predominant of complexes formation, while dynamic collision could be negligible.

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3.4. Binding parameters The binding constant (K) and the number of binding sites between docetaxel with HSA can be calculated using the equation for the static quenching process [20].   F0  F log ¼ log K þ n log½Q  ð3Þ F A plot of log [(F0–F)/F] versus log [Q] gives a straight line, whose slope equals to n and the intercept on Y-axis equals to log K. The values of K and n at 283, 300 and 310 K are listed in Table 1. The fact that the binding constant between docetaxel and HSA decreased with rising temperature, which implies that the stability of the HSA–docetaxel complex was weakened with rising temperature. Furthermore, the value for n close to one, we may infer that there was only one docetaxel binding site which most likely resided in the hydrophobic pocket of HSA. 3.5. Thermodynamic analysis for binding mode between docetaxel and HSA There are four major noncovalent interaction forces between small molecules and proteins: hydrogen bonding, van der Waals force, hydrophobic interaction and electrostatic interaction [23]. The signs and magnitudes of thermodynamic parameters for protein reactions account for the main forces contributing to protein stability. If the enthalpy change (DH0) does not vary significantly over the temperature range studied, then the thermodynamic parameter of DH0, entropy change (DS0) and free energy (DG0) can be determined from the Van’t Hoff equation log K ¼ 

DH0 2:303RT

þ

DS0

0

The results of DH0, DS0 and DG0 are presented in Table 2. The negative DG0 means that the binding process was spontaneous and the formation of docetaxel–HSA complex was an exothermic reaction accompanied with negative DH0.The major contribution to DG0 arising from the DH0 term rather than from DS0 implies that the binding processes were enthalpy driven. Ross and Subramanian [24] have characterized the sign and magnitude of the thermodynamic parameter associated with various individual kinds of interactions that may take place in protein association processes. From the viewpoint of water structure, specific electrostatic interactions between ionic species in aqueous solution are characterized by a negative DH0 and positive DS0 values, while the negative DH0 and negative DS0 values, which are considered as typical evidence for hydrogen bonding and van der Waals forces in low dielectric media [24,25]. According to experiment results, the hydrogen bond played major role in binding of docetaxel to HSA. In addition, under the experiment conditions here (pH 7.4) docetaxel might be considered to be partly ionized according to its structure. Krisztina Paal et al. [26] find, by molecular docking, there are many ionic and polar residues in proximity of the docetaxel. Hence, the electrostatic interactions may also contribute to the binding.

3.6. ANS-binding experiment 1-anilino-8-naphthalenesulfonate is often used to detect the presence of hydrophobic regions of protein [27–29]. Fig. 6 shows, with ANS alone, less fluorescence emission was detected. In contrast, when ANS bound to HSA, the fluorescence strength increased remarkably. Furthermore, the fluorescence quenching experiment of HSA–ANS was designed to find possible binding site of docetaxel on HSA. As shown in Fig. 7, it probably indicates that

ð4Þ

2:303R

where K is the binding constant at the corresponding temperature and R is the gas constant. The DH0 and DS0 are determined from the linear Van’t Hoff plots. The linearity of the ln K versus 1/T plots is shown in Fig. 5 The DG0 is estimated from the following equation: 0

1199

0

DG ¼ DH  T DS

Table 2 Thermodynamic parameters of HSA–docetaxel interaction. T (K)

K (  104 l mol1)

DG0

DH0 (KJ mol1)

DS0 (J mol1 K1)

283 300 310

9.80 3.38 2.18

27.00 26.15 25.66

41.07

49.72

ð5Þ

10000

11.6

Fluorescence Intensity

11.4 11.2

10.8 10.6 10.4

9 6000 1

4000

2000

10.2 10.0

0

9.8

5

450

500 Wavelength (nm)

550

600

0.

00

35

50 00 3

5 0.

00

34

0 0.

00

34

5 0.

0

00 33 0.

00

33

5 0.

0.

00

32

0

400

32 00 0.

lnK

11.0

8000

1/T (K-1) Fig. 5. Van’t Hoff plot for the interaction of HSA and docetaxel, pH ¼ 7.4.

Fig. 6. Fluorescence spectra of ANS in presence of HSA. Concentration of ANS: 10 X106 mol l1; 1–9 concentration of HSA: (a) 0; (b) 1 106 mol l1; (c) 2  106 mol l1; (d) 3  106 mol l1; (e) 4  106 mol l1; (f) 5  106 mol l1; (g) 6  106 mol l1; (h) 7  106 mol l1; and (i) 8  106 mol l1.

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3500 1

a 4000

2500

11

Fluorensence Intensity

Fluorescence Intensity

3000

2000 1500 1000 500

h

3000

2000

1000

0 400

450

500 Wavelength (nm)

550

0

600

285

270

Fig. 7. Fluorescence spectra of ANS–HSA in presence of docetaxel. 1–11 concentration of docetaxel: (a) 0; (b) 4  106 mol l1; (c) 8  106 mol l1; (d) 12  106 mol l1; (e) 16  106 mol l1; (f) 20  106 mol l1; (g) 24  106 mol l1; (h) 28  106 mol l1; (i)  106 mol l1; (j) 36  106 mol l1; and (k) 40  106 mol l1.

300 315 Wavelength (nm)

3000

330

a

docetaxel replaced ANS and connected with HSA. Docetaxel and ANS probably had same bonding site with HSA. Because ANS bound to the hydrophobic cavity of HSA, docetaxel also bound to the hydrophobic cavity of HSA. These results were in according with the results of our Binding parameters and further confirmed by the synchronous fluorescence spectra described below.

Fluorescence Intensity

2500

3.7. Conformation investigation

1500 1000 500 0 260

280

300

320

340

360

380

400

Wavelength (nm) Fig. 8. The synchronous fluorescence of HSA–docetaxel system at 298 K (a) Dl ¼ 15 nm and (b) Dl ¼ 60 nm. The concentration of docetaxel (a) 0; (b) 1 106 mol l1; (c) 2  106 mol l1; (d) 3  106 mol l1; (e) 4  106 mol l1; (f) 5  106 mol l1; (g) 6  106 mol l1; and (h) 7  106 mol l1; The concentration of HSA is 2.0  106 mol l1, pH 7.4.

0 a -5000 [θ] (deg.cm2.dmol-1)

The synchronous fluorescence spectra give information about the molecular environment in the vicinity of the chromospheres molecules. The fluorescence of HSA come form tyrosine, tryptophan and phenylalanine residues. According to Yuan et al. [30], when the D-value (Dl) between excitation wavelength and emission wavelength are stable at 60 nm, the synchronous fluorescence gives the characteristic information of tryptophan residues. When Dl value is 15 nm, the synchronous fluorescence gives the characteristic information of tyrosine residue. The effect of docetaxel on HSA synchronous fluorescence spectroscopy is shown in Fig. 8. The position of the maximum emission wavelength had a small red shift (from 337 to 338 nm) when Dl was 60 nm, while the position of the maximum emission wavelength did not change when Dl was 15 nm. It is reported that the maximum emission wavelength at 330–332 nm shows that tryptophan residues are located in the nonpolar regions, which means they are buried in a hydrophobic cavity in HSA; the maximum emission wavelength at 350–352 nm shows that tryptophan residues are exposed to water, namely the hydrophobic cavity in HSA is disagglomerated and the structure of HSA getting loose [20]. The results infer that docetaxel bound in hydrophobic cavity of HSA and the polarity around the tryptophan residues increased slightly [31]. It is also likely that tryptophan residue is closer to docetaxel than tyrosine residue. The study on CD was further performed to find possible influence of docetaxel to the secondary structure of HSA (Fig. 9). CD spectra of HSA exhibit two negative bands in the ultraviolet region at 209 and 222 nm, which is characteristic for a-helical structure of protein. The reasonable explanation is that the negative peaks between 208 and 209 nm and 222–223 nm are both contributed to n-p* transfer for the peptide bond of

h

2000

c

-10000

-15000

-20000 200

210

220 230 240 Wavelength (nm)

250

260

Fig. 9. CD spectra of HSA in the presence of docetaxel at 298 K. Concentration of docetaxel: (a) 0; (b) 12  106 mol l1; (c) 24  106 mol l1; and concentration of HSA is 2.0  106 mol l1.

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absence of docetaxel are similar in shape indicates that the structure of HSA was also predominantly a-helical. The binding of docetaxel to HSA caused increase in both two bands, indicating the increase of the a-helical content in proteins. The CD results were expressed in terms of mean residue ellipticity (MRE) in deg cm2 dmol1 according to the following equation [33]: MRE ¼

observed CDðm degÞ Cp nl  10

ð6Þ

where Cp is the molar concentration of the protein, n the number of amino acid residues of the protein and l the path length. The a-helical content of HSA is calculated from MRE value at 209 nm, using the following equation [34]:

a  helixð%Þ ¼

MRE209  4000  100 33; 000  4000

ð7Þ

where MRE209 is the observed MRE value at 209 nm, 4000 the MRE of the b-form and random coil conformation cross at 209 nm, 33,000 is the MRE value of a pure a-helix at 209 nm. From the above equation, the a-helical structure of HSA is increased from 48.73% to 50.32% and 52.64% at the molar ration ratio of HSA to docetaxel of 1:6 and 1:12. The percentage of a-helical structure in HSA increased indicates that docetaxel strengthened their hydrogen bonding networks and stabilized helical structures in protein upon the addition of docetaxel [35,36]. These results further verify hydrogen bonds play a major role in binding and are consistent with the thermodynamic analysis. In order to obtain more information on the binding of docetaxel to HSA, the FT-IR spectroscopy was investigated. In IR region, the frequencies of bands due to the amide I and amide II are sensitive to the secondary structure of protein. Particularly, the amide I band is useful for the secondary studies. The protein amide I occurs in the region 1600–1700 cm1 (mainly CQO stretching vibrations of amide groups) and the amide II is in the region 1500–1600 cm1 (mainly C–N stretch couple with N–H bending model). Amide I band is more sensitive to the change of protein secondary structure than amide II [37]. Fig. 10 shows that the peak position of amide I and amide II shifted from 1645.9 to

1643.9 cm1 and 1566.3 to 1550.7 cm1. The absorption of amide I and II both increased with docetaxel added. These indicate that the secondary structure of the protein has been changed. The docetaxel interacts with the CQO and C–N groups in the protein polypeptides and causes the rearrangement of the polypeptide carbonyl hydrogen-bonding network. According to the wellestablished assignment criterion, the spectral ranges from 1615 to 1637 cm1, 1638–1648 cm1, 1649–1660 cm1, 1660–1680 cm1 and 1680–1692 cm1 in the amide I are attributed to b-sheet, random coil, a-helix, b-turn and b-antiparallel structures, respectively. The quantitative analysis of the protein secondary structure for free HSA and docetaxel–HAS complex was given in Fig. 11 and Table 3. The curve-fitted results show that the a-helix increased from 43.23% to 47.99%, b-sheet increased from 19.66% to 20.69% and b-turn increased from 15.16% to 16.17%. The tendency of increasing of the a-helix is consistent with the CD results. From the CD and FT-IR results, it is apparent that the binding of docetaxel to HSA causes a conformational change of the protein. The contents of the secondary structures determined by CD and FT-IR differ from each other, although the trend of changes is similar. The major cause for the difference may lie in the spectroscopic signals themselves: FT-IR signals arise from the

1650.2

Absorbance

a-helical [32]. The fact that CD spectra of HSA in the presence or

1201

1667.9

1630.6 1640.8

1682.6

1614.3

1600

1620

0.014

1640 1660 Wavelength (cm-1)

1680

1700

amide I 1643.9

0.012

Absorbance

1645.9

0.008 0.006

0.002 0.000 1500

amide II 1550.7

0.004

b

1556.3

Absorbance

0.010

1550

1649.8

1624.1

a

1668.0 1640.2

1600

1650

1700

1750

Wavelength (cm-1) Fig. 10. FT-IR spectra of HSA in PBS of pH 7.4: (a) FT-IR spectra of HSA (subtracting the absorption of the buffer solution from the spectrum of the protein solution); (b) FT-IR difference spectra of HSA (subtracting the absorption of the docetaxel free form from that of the docetaxel–HSA complex). CHSA ¼ 2  105 mol l1 and Cdocetaxel ¼ 1.2  104 mol l1.

1683.7

1608.2

1600

1620

1640 1660 Wavelength (cm-1)

1680

1700

Fig. 11. The curve-fitting amide I region (1600–1700 cm1) with secondary structure determination of free HSA (a) and docetaxel–HSA complex (b) in PBS of pH 7.4, CHSA/Cdocetaxel ¼ 1:6.

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Table 3 Secondary structure determination for the free HSA and docetaxel–HSA in PBS (pH ¼ 7.4) at the molar concentration ration CHSA/Cdocetaxel ¼ 1:6. Components

a-helix (%)

b-sheet (%)

b-turn (%)

FT-IR

Free-HSA HSA–docetaxel

43.23 47.99

19.66 20.69

15.16 16.17

CD

Free-HSA HSA–docetaxel

48.73 50.32

and F ¼ 0.1118 [39]. Hence, from equations, the following R0 ¼ 2.58 nm and parameters J ¼ 1.38  1014 cm3 l mol1, r ¼ 4.72 nm were calculated. The donor to acceptor distance is less than 7 nm [40], which indicates the energy transfer from HSA to docetaxel occurred with high probability. Larger HSA–docetaxel distance, r compared to that of R0 value observed also reveals the static-type quenching mechanism to a large extent [41]. The overlap of the absorption spectrum of docetaxel and fluorescence emission spectra of HSA is shown in Fig. 12.

4. Conclusions

0.25

a

2000

0.20

1500

0.15

1000

0.10

Absorbance

Fluorescence Intensity

2500

0.05

500 b 0 350

400 450 wavelength (nm)

0.00 500

Fig. 12. The overlap of the fluorescence spectrum of HSA (a) and the absorbance spectrum of docetaxel (b).

This paper provides an approach study the binding of docetaxel to HSA by employing difference optical techniques. The studies have indicated that docetaxel is a quencher for HSA and quenching mechanism is a static quenching procedure. Our results show that the interaction between docetaxel and HSA induces a conformational change of HSA, which is further proved by the quantitative analysis data of UV–vis, CD, FT-IR and fluorescence spectrum. Based on the binding parameters and thermodynamic analysis, it can be found that there is only one docetaxel binding site on HSA, binding reaction is spontaneous and both hydrogen bond and electrostatic interactions contribute to the binding. The ANS-binding experiments and synchronous fluorescence spectra further verify bonding site residing in hydrophobic regions of HSA and the important role of hydrogen bond in stabilizing docetaxel–HSA complex. The results obtained here by using spectrophotometer techniques are scientific. We hope our study may shed light on transportation of docetaxel in vivo. This work can also provide some important information to clinical research and the theoretical basis for new drug designing and new dosage form development.

vibration, whereas CD spectra are obtained from electronic transitions. Acknowledgements 3.8. The energy transfer between docetaxel and HSA Energy transfer phenomena have wide applications in energy ¨ conversion processes. According to the Forster’s non-radiative energy transfer theory [38], if the emitted fluorescence from a donor could be absorbed by an acceptor, energy may transfer from the donor to the acceptor. The efficiency of energy transfer (E) is related to the distance R between donor and acceptor by R60 F ¼ E¼1 F0 R60 þ r 6

ð8Þ

where F and F0 are the fluorescence intensities of HSA in presence and absence of docetaxel; r the binding distance between the donor and acceptor; and R0 the critical distance between the donor and acceptor when their transfer efficiency is 50%. R60 ¼ 8:8  1025 k2 N4 FJ

ð9Þ

2

where k is the spatial orientation factor of the dipole, N the refractive index of the medium, F the fluorescence quantum yield of the donor and J the overlap integral of the fluorescence emission spectrum of the donor with the absorption spectrum of the acceptor, which can be calculated by the equation J¼

X

l4 Dl FðlÞDl

FðlÞeðlÞ P

ð10Þ

where F (l) is the fluorescence intensity of the fluorescent donor at wavelength l and e (l) the molar absorption coefficient of the acceptor at wavelength l. In the present case, k2 ¼ 2/3, N ¼ 1.336

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