Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 786–794

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Study on the interaction between bovine serum albumin and 40 -azido-20 -deoxyfluoroarabinocytidine or analogs by spectroscopy and molecular modeling Ruiyong Wang ⇑, Xiaogai Wang, Zhigang Li, Yuanzhe Xie, Lingling Yang, Jie Shi, Junbiao Chang ⇑ College of Chemistry and Molecular Engineering, Zhengzhou University, 100 Science Avenue, Zhengzhou 450001, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The interactions between BSA and

The fluorescence spectra of BSA in the absence and presence of FNC. 1 800

F lu o re s c e n c e In te n s ity

FNC or analogs have been investigated.  Hydrophobic interactions play major role in the binding process.  The influence of molecular structure on the binding aspects has been investigated.  Molecular docking was also applied in the binding study.

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W a ve le n g th (n m )

a r t i c l e

i n f o

Article history: Received 17 April 2014 Received in revised form 12 May 2014 Accepted 25 May 2014 Available online 11 June 2014 Keywords: Bovine serum albumin FNC Analogs Spectroscopy

a b s t r a c t The binding of 40 -azido-20 -deoxyfluoroarabinocytidine (FNC) or analogs (cytidine and 50 -cytidylate monophosphate) to bovine serum albumin (BSA) was investigated by fluorescence, UV–vis absorption spectroscopy and molecular modeling. The three compounds quenched the intrinsic fluorescence of BSA and the results revealed the presence of static quenching mechanism. The positive DH and positive DS for the systems suggested that the hydrophobic forces stabilized the interaction between the compounds and protein. Results also showed that FNC was the weakest quencher. Ó 2014 Elsevier B.V. All rights reserved.

Introduction Proteins are the most abundant macromolecules in cells and are crucial to maintaining normal cell functions. The serum albumin, one of the most abundant proteins, plays an important role in ⇑ Corresponding authors. Tel.: +86 371 67781588. E-mail addresses: [email protected] (R. Wang), [email protected] (J. Chang). http://dx.doi.org/10.1016/j.saa.2014.05.090 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

the transport and deposition of a variety of endogenous and exogenous ligands in blood [1]. In this paper, bovine serum albumin (BSA) was selected as the protein model because of its low cost, ready availability, and unusual ligand-binding properties. BSA is constituted by 582 amino acid residues. On the basis of the distribution of the disulfide bridges and of the amino acid sequence it seems possible to regard BSA as composed of three linearly arranged, structurally distinct, and evolutionarily related domains I, II and III. Further, each domain is subdivided into two

R. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 786–794

Apparatus

NH2

Fluorescence spectra were carried out on a 970-CRT Spectrofluorimeter (San Ke, Shanghai, China) equipped with a 1 cm quartz cell and a thermostat bath. The excitation and emission slit widths were set at 5.0 nm. Absorption spectra were acquired in an Agilent 8453 UV–visible spectrophotometer. The pH values were measured by a pH-3 digital pH-meter (Lei Ci, Shanghai) with a combined glass electrode.

N

O HO

N3

N

787

O F

HO

FNC

5'-CMP

CDPC

Fig. 1. The structure of 40 -azido-20 -deoxyfluoroarabinocytidine or 20 -deoxy-20 -bfluoro-40 -azidocytidine (FNC), Cytidine (CDPC) and 50 -cytidylate monophosphate (50 -CMP).

subdomains A and B. Meanwhile, BSA has two tryptophan residues that possess intrinsic fluorescence. Trp-212 is located within a hydrophobic binding pocket of the protein and Trp-134 is located on the surface of the molecule [2]. Human cancer remains a major public health problem worldwide, and right now, tremendous effort has been directed to the discovery and development of novel agents for the treatment of human cancer and great successes have been achieved. So far, several nucleoside analogues have been successfully used as anticancer drugs. 40 -azido-20 -deoxyfluoroarabinocytidine or 20 -deoxy-20 -b-fluoro-40 azidocytidine (FNC, Fig. 1) is a novel cytidine analogue [3]. Recent studies have demonstrated that FNC is a highly potent and selective deoxycytidine inhibitor, which has anticancer activity and been found to suppress the secretion of the HBV antigens in a dose-dependent manner and hepatitis C virus (HCV) [4,5]. Cytidine (CDPC, Fig. 1) is one of the pyrimidine nucleoside constitute a nucleic acid, which is mainly used for acute craniocerebral trauma and disturbance of consciousness after brain surgery. Cytidine monophosphate (50 -CMP, Fig. 1) is a structural portion of RNA, which can be used as production of nucleic acid drugs intermediates, health food, biochemical reagents and biochemical drugs. Study of drug–protein interaction has great significance in discovering pharmacokinetic and pharmaco-dynamics implications, which is an essential step for a new drug design. Investigation of the interaction is helpful for revealing the transportation and distribution of the drugs in vivo, explaining the toxicity at the molecular level [6]. Fluorescence assay has been widely applied to study the interaction because of some advantages such as high sensitivity, high selectivity and easy operation. In this work, for the first time the interaction between BSA and FNC (analogs) has been studied by fluorescence spectroscopy, UV–vis absorption spectroscopy and molecular modeling method.

Experimental Fluorescence measurement In titration experiments, the concentration of protein was kept fixed at 8.00  106 mol L1 while that of FNC or analogs (CDPC and 50 -CMP) were varied from 0 to 3.2  105 mol L1 in a total volume of 2.0 mL. Fluorescence spectra were recorded in the range of 300–450 nm for BSA (kex = 290 nm). Quenching experiments were carried out at 302 and 317 K, respectively. Synchronous fluorescence spectra were recorded with Dk = 15 nm and 60 nm (Dk = kem–kex) in the absence and presence of compounds and the spectra were recorded in the range of 280–400 nm. The three-dimensional fluorescence spectra of BSA were recorded by scanning excitation wavelength in the range of 230–305 nm, and emission wavelength in the range of 280–460 nm at an interval of 5 nm, respectively. Absorbance measurements The UV–vis absorption spectra of BSA, FNC (or analogs) and their mixture were obtained in the range of 190–350 nm at room temperature, respectively. Molecular modeling study The PDB entry of the BSA crystal structure employed in docking study was 1H9Z. The structure of FNC, CDPC and 50 -CMP was optimized using Gaussian 09 program [7]. Molecular docking simulations were performed with the software package AutoDock4.2 [8], in which the Lamarckian Genetic Algorithm was applied. The program was used to calculate the interaction modes between FNC (CDPC or 5-CMP) and BSA. Geometry optimizations were carried out using the hybrid B3LYP [9] functional together with the 6–31 g (d, p) basis set for C, H, O, F and N atoms. A grid map of 126  78  126 grid points in size with a grid-points pacing of 0.708 Å was created for the two proteins. According to the binding energy and the geometry matching after 250 runs, the most favorable docking model was selected for further analysis. Results and discussion

Experimental section

Fluorescence quenching spectra

Materials

Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interaction which including excited state reactions, molecular rearrangements, energy transfer, ground state complex formation, and collisional quenching. Such decrease in intensity of fluorescence is called fluorescence quenching. For macromolecules, the fluorescence measurements can give some information of the binding of small molecule substances to proteins, such as the binding mechanism, binding mode, binding constants, binding sites, and intermolecular distances [10]. The fluorescence emission spectra of BSA in the presence of FNC or analogs at 302 K were shown in Fig. 2. BSA had a strong fluorescence emission band with a peak at 342 nm by fixing the excitation

BSA (Fraction V, heatshock isolation, >99%) was obtained from Sigma–Aldrich. FNC was synthesized in our lab. Both CDPC (99%) and 50 -CMP (98%) were obtained from Aladdin Chemistry Co., Ltd. BSA was directly dissolved in double distilled water to prepare stock solutions (1.0  104 mol L1) which were then stored at 0– 4 °C. Stock solutions (0.02 mol L1) of FNC or analogs (CDPC and 50 CMP) were dissolved in double distilled water. 0.5 mol L1 NaCl solution was used to keep the ion strength of solutions. 0.1 mol L1 Tris–HCl buffer was selected to keep the pH of the solution at 7.40. All chemicals were of analytical grade and were used without further purification. Double-distilled water was used throughout.

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wavelength at 290 nm. When different amount of compounds was titrated into a fixed concentration of BSA, the fluorescence intensity of BSA decreased regularly with no shift of the emission wavelength. Results suggested that the three compounds might interact with BSA. Fluorescence quenching mechanisms and binding constants Fluorescence quenching refers to any process that decreases the fluorescence intensity of a sample. It is usually classified into either dynamic or static quenching, which can be distinguished by their differing dependence on temperature and viscosity [11]. Dynamic

a

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Fluorescence Intensity

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500 400 300 200 100 0 300

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Wavelength (nm) 900

2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9

a

317K FNC CDPC 5'-CMP

-5

0

5

2.4

700

15 -4

25

30

35

25

30

35

m o l/L

FNC CDPC 5'-CMP

2.0

500

20

b 302K

2.2

1

600

F 0 /F

9

400 300 200

1.8 1.6 1.4

100 0 300

10

[Q ]/1 0

c

800

Fluorescence Intensity

ð2Þ

Fig. 3 displayed the Stern–Volmer plots of the quenching of BSA fluorescence by three compounds at different temperatures (302 and 317 K). The calculated KSV and kq values were presented in Table 1. There was a linear dependence between F0/F and [Q]. The maximum scatter collision quenching constant of various quenchers with biopolymer is 2.0  1010 L mol1 s1 [15]. The rate constants of quenching for FNC (CDPC or 50 -CMP) were greater than the maximum value of kq for the scatter mechanism. This indicated that the static quenching may also exist between them [16].

b

700

ð1Þ

where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. kq is the bimolecular quenching rate constant, s0 is the lifetime of the fluorescence in the absence of quencher (for most bimolecular, s0 is about 108 s) [14], [Q] is the concentration of quencher and KSV is the Stern–Volmer quenching constant. The value of kq is deduced by the following equation:

F 0 /F

Fluorescence Intensity

800

F0 ¼ 1 þ K SV ½Q ¼ 1 þ kq s0 ½Q  F

kq ¼ K SV =s0

600 500

quenching depends upon diffusion. Because higher temperatures result in larger diffusion coefficients, the biomolecular quenching constants are expected to increase with increasing temperature. In contrast, increased temperature is likely to result in decreasing stability of complexes and, thus, lower values of the static quenching constants [12]. In order to confirm the quenching mechanism, the well known Stern–Volmer equation is used for analyzing the fluorescence quenching data [13]:

1.2 320

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Wavelength (nm) Fig. 2. Effect of FNC (a) or analogs (CDPC for b and 50 -CMP for c) on fluorescence spectra of BSA (T = 302 K, pH = 7.40, kex = 290 nm). The concentration of BSA was fixed at 8.00  106 mol L1 while the concentration of FNC (analogs) corresponding to 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2  104 mol L1, respectively (From spectra 1 to 9).

1.0 -5

0

5

10

15 -4

[Q ]/1 0

20

mol/L

Fig. 3. The Stern–Volmer curves for the quenching of BSA with FNC (analogs) at two temperatures (302 K and 317 K). Other experimental conditions were the same as those in Fig. 2.

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R. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 786–794 Table 1 Stern–Volmer quenching constants of the system at different temperatures. Compound

T (K)

KSV (L mol1)

Stern–Volmer equation 3

kq (L mol1 s1)

3

11

R

FNC

302 317 302

F0/F = 0.9749 + 2.706  10 [Q] F0/F = 0.9945 + 2.352  103 [Q] F0/F = 0.9544 + 3.921  103 [Q]

2.706  10 2.352  103 3.921  103

2.706  10 2.352  1011 3.921  1011

0.9975 0.9978 0.9977

CDPC

317 302

F0/F = 0.9717 + 3.318  103 [Q] F0/F = 0.9540 + 3.858  103 [Q]

3.318  103 3.858  103

3.318  1011 3.858  1011

0.9978 0.9978

50 -CMP

302

F0/F = 0.9697 + 3.484  103 [Q]

3.484  103

3.484  1011

0.9981

3.0

For static quenching, the absorption spectra of fluorescence substance change because of the formation of ground-state complex. In contrast, the dynamic quenching only affects the excited states of the fluorophore and thus no changes in the absorption spectra are expected [17]. The absorption spectral change of BSA in the absence and presence of the three compounds was shown in Fig. 4. By comparing the curve 1 with curve 4 in Fig. 4, the absorbance of BSA decreased with the addition of FNC (analogs). The results indicated that the static quenching existed in the binding process, which might cause the slight change of the conformation of protein [18].

a

Absorbance

2.5 2.0 1 2 3 4

1.5 1.0 0.5

Binding parameters

0.0 200

250

300

350

Wavelength (nm) 3.0

For static quenching, the binding constants (Ka) and the number sites (n) can be determined by the following equation [19]:

b 0.2 a

0.0

2.0 1.5

lo g [(F 0 -F )/F ]

Absorbance

2.5

1 2 3 4

1.0 0.5 0.0

317K FN C CDPC 5 '-C M P

-0.2 -0.4 -0.6 -0.8

200

250

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350 -1.0

Wavelength (nm)

-4.4 3.0

-4.0

-3.8

-3.6

-3.4

-3.8

-3.6

-3.4

log [Q]

c 0.2

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2.0 1 2 3 4

1.5 1.0

lo g [(F 0 -F )/F ]

Absorbance

-4.2

0.5 0.0 200

250

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350

Wavelength (nm) Fig. 4. Absorption spectra of FNC (a) or analogs (CDPC for b and 50 -CMP for c) bound to BSA at pH 7.40. (1) absorption spectra of BSA only, the concentration of BSA was 8.00  106 mol L1; (2) absorption spectra of FNC or analogs only, the concentration of FNC (analogs) was 8.00  106 mol L1; (3) absorption spectra of BSA + FNC or its analogs system; (4) absorption spectra of [BSA + FNC or analogs] – [FNC or analogs].

b

302K FN C CDPC 5 '-C M P

-0.2 -0.4 -0.6 -0.8 -1.0 -1.2 -4.4

-4.2

-4.0

log [Q] Fig. 5. Plots of log(F0–F)/F versus log [Q] for the quenching of FNC or analogs (CDPC and 50 -CMP) with BSA at two temperatures (302 K and 317 K). Other experimental conditions were the same as those in Fig. 2.

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Table 2 Thermodynamic parameters of the interaction between FNC (analogs) to BSA.

3

n

R

DH0 (kJ mol1)

DG0 (kJ mol1)

DS0 (J mol1 K1)

FNC

317 302 317

1.950  10 5.082  103 4.955  103

0.9804 1.082 1.055

0.9977 0.9993 0.9982

50.83

19.10 22.49 21.36

231.6

CDPC

302 317

1.099  104 6.012  103

1.131 1.072

0.9995 0.9990

42.27

24.52 21.85

210.7

50 -CMP

302

1.148  104

1.138

0.9998

34.32

24.64

186.0

log

F0  F ¼ log K a þ n log½Q  F

900

The value of Ka and n were obtained from the slope of double logarithm regression curve of log(F0–F)/F versus log[Q] (Fig. 5) and the corresponding values were given in Table 2. The binding constants increased with rising temperatures, which suggested that the binding reaction between the three compounds and BSA was endothermic [20]. The binding constants of the interaction between the three compounds and BSA increased in the following order: FNC < CDPC < 50 -CMP. The value of n was approximately equal to 1. Results also showed that FNC was the weakest quencher.

800

Fluorescence Intensity

ð3Þ

600 0.006

500 400

Energy transfer According to Förster’s non-radioactive energy transfer theory (FRET) [23], FRET is a special type of dynamic quenching mechanism where exists distance-dependent interaction between the

0.004

A

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0.004 A

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0.002

100 0 300

ð5Þ

350

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450

0.000

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900 800 c

Fluorescence Intensity

where R is the gas constant, and T is the temperature, KA is binding constant at the corresponding temperature. As presented in Table 2, DG was found negative, both DS and DH were positive for the three systems. The negative values of DG revealed that the binding of the three compounds to BSA was a spontaneous process [21]. From the thermodynamic standpoint, DH > 0 and DS > 0 implies a hydrophobic interaction; DH < 0 and DS < 0 reflects the Van der Waals force or hydrogen bond formation; DH  0 and DS > 0 suggests an electrostatic force [22]. The positive values of DH and DS observed in the present study revealed the presence of hydrophobic forces. BSA has two tryptophan residues. Trp-214 is located in a hydrophobic fold and the additional tryptophan (Trp-135) is located on the surface of the molecule. In this paper, FNC or analogs was probably bound to Trp-214 residue mainly through hydrophobic interaction according to the thermodynamic results.

0.008

Wavelength (nm)

Fluorescence Intensity

ð4Þ

the values of entropy change (DS) and the free energy change (DG) can be calculated from the Van’t Hoff equation:

DG ¼ DH  T DS ¼ RT ln K A

F

700

0 300

The thermodynamic parameters, enthalpy change (DH) and entropy change (DS) of binding reaction are the main evidence for confirming binding modes. The temperatures were chosen at 302 and 317 K, and thus BSA will not undergo any structural degradation. Because the temperature effect is small, the reaction enthalpy change can be regard as a constant if the temperature range is not too wide. The thermodynamic parameters could be calculated according to be determined from the equation below:

  K A2 DH 1 1 ¼  R T2 T1 K A1

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100

Binding mode

ln

0.010

Absorbance

Ka (L mol1)

Absorbance

T (K)

F

0.008

700 600

0.006

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A

0.004

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Absorbance

Compound

0.002

100 0 300

350

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0.000

Wavelength (nm) Fig. 6. The overlap of the UV absorption spectrum of FNC (a) or analogs (CDPC for (b) and 50 -CMP for (c)) with the fluorescence emission spectrum of BSA. Curve F is the fluorescence spectrum of BSA (8.00  106 mol L1), curve A is the UV absorbance spectrum of FNC (analogs) (8.00  106 mol L1). (T = 302 K, pH = 7.40).

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R. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 786–794 Table 3 Energy transfer parameters in the system. J (cm3 L mol1)

Compound

16

FNC CDPC 50 -CMP

6.965  10 5.451  1016 7.635  1016

E

R0 (nm)

r (nm)

0.04629 0.05382 0.05556

1.64 1.57 1.66

2.71 2.53 2.66

E¼1

electronic excited states of two fluorophores in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. Energy transfer is likely to happen under the following conditions: (1) the relative orientation of the donor and acceptor dipoles, (2) the extent of overlap of fluorescence emission spectrum of the donor with the absorption spectrum of

180

the acceptor, and (3) the distance between the donor and the acceptor is less than 7 nm. The efficiency of energy transfer (E) is related to the distance R between donor and acceptor by following equation [24]:

F R6 ¼ 6 0 F 0 R0 þ r 60

ð6Þ

where r is the binding distance between donor and receptor and R0 is the critical distance at which transfer efficiency equals to 50%. It can be calculated from donor emission and acceptor absorption spectra using the Förster’s formula:

R60 ¼ 8:79  1025 K 2 n4 UJ

ð7Þ

900

a

800

FNC

160

a'

700

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1

IF

IF

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IF

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Fig. 7. The synchronous fluorescence spectra of BSA in the absence and presence of FNC (analogs) while the Dk = 15 nm (a, b and c) and Dk = 60 nm (a0 , b0 and c0 ). The concentration of BSA was fixed at 8.00  106 mol L1 while the concentration of FNC (analogs) corresponding to 0, 0.4, 0.8, 1.2, 1.6  104 mol L1, respectively (From spectra 1 to 5). Other experimental conditions were the same as described in Fig. 2.

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where K2 is the orientation factor related to the geometry of the donor and acceptor of dipoles, and K2 is for random orientation as in fluid solution; n is the refractive index of the medium, U is the fluorescence quantum yield of the donor, and J is the overlap integral of the fluorescence emission spectrum of the donor with the absorption spectrum of the acceptor, which can be calculated by the following equation:

R1 J¼

0

FðkÞeðkÞk4 dk R1 FðkÞdk 0

ð8Þ

where F(k) is the fluorescence intensity of the fluorescent donor at wavelength k, and e(k) is the molar absorption coefficient of the

acceptor at wavelength k. Fig. 6 showed the spectral overlap between absorption spectrum of the three compounds and fluorescence spectrum of BSA in the wavelength range of 300–500 nm. It was reported that K2 = 2/3, U = 0.15 and n = 1.336 for BSA [25]. The calculated results of J, R0, E and r were listed Table 3. It was observed that the distance between BSA and FNC (analogs) was less than 7 nm [26]. The distance between the Trp residue (as donor) and the interacted compounds (as acceptor) is 2–8 nm scale with the rule 0.5R0 < r < 2.0R0 [27]. The results indicated that the energy transfer from BSA to FNC (analogs) occurred with high probability [26].

Fig. 8. The docking results of FNC (a and a0 ) or analogs (CDPC for b and b0 , 50 -CMP for c and c0 ) with BSA systems. In a, b and c, the serum albumin was represented using solid ribbon and the ligand structure is represented using ball-stick model. In a0 , b0 and c0 , the residues of BSA and the structure of FNC or analogs (CDPC and 50 -CMP) were represented using stick model.

R. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 786–794

Synchronous fluorescence spectra As well known, synchronous fluorescence spectra can provide the information on the molecular microenvironment, particularly in the vicinity of the fluorophore functional groups [28]. It is an ideal tool to investigate effects of drugs on the conformation of protein. When the wavelength intervals (Dk) are stabilize at 15 or 60 nm, the synchronous fluorescence gives the characteristic information of tyrosine residues or tryptophan residues, respectively [26]. The shifts in the position of the synchronous maxima of these residues (which usually occur upon binding) give indication on the changes in polarity around these particular fluoroscope and thus on proximity of the ligand [29]. The effect of FNC (analogs) on BSA synchronous fluorescence spectra was shown in Fig. 7. It was apparent that the intensities decreased with the addition of FNC for both tyrosine and tryptophan. In Fig. 7a–c there were a significant red-shift on the spectra of tyrosine with a slender decrease of the intensity after addition of compounds. Both slight red-shift and rapid decrease of the intensity was observed in Fig. 7. The observed red shift indicated a transition of tyrosine and tryptophan residues in BSA from a nonpolar to a more polar environment, and more residues expose to hydrophilic and polar micro-environment. Results showed that the binding of FNC or analogs to BSA resulted in microenvironmental and conformational changes in BSA.

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was in agreement with the binding mode proposed in thermodynamic analysis. Conclusions In summary, the interactions between FNC (analogs) and BSA under physiological conditions were studied by spectroscopic techniques in combination with molecular modeling. Fluorescence spectroscopy provides qualitative and quantitative information about the interaction between FNC (analogs) and BSA. Our results showed that the intrinsic fluorescence of BSA was quenched by FNC (analogs). The binding reaction mainly involved hydrophobic force as revealed by thermodynamic parameters. Changes in the microenvironment of the protein were also indicated by synchronous fluorescence and three-dimensional spectra. Results showed that FNC was the weakest quencher. The binding study of drugs to proteins is important in pharmacy, pharmacology and biochemistry and so on. Acknowledgements We are grateful to the National Natural Science Foundation of China (Nos. 81330075 and 21172202), and 2013 Key science and technology plan project of Henan province (132102110051) for financial support.

Three-dimensional fluorescence spectroscopy

Appendix A. Supplementary material

It is well-known that three-dimensional fluorescence and the contour spectra can provide more detailed information about the configuration of proteins. The contour map displayed a bird’s eye view of the fluorescence spectra. The three-dimensional fluorescence contour spectra of BSA (a), BSA + FNC or analogs (b, c, d) and the corresponding projections spectra of (a0 , b0 , c0 , d0 ) were shown in Supporting information. Result showed that the intensity of the contour map of BSA decreased obviously but to different degrees.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.05.090.

Molecular modeling The complementary applications of molecule modeling have been employed by computer methods to improve the understanding of the interactions. Descriptions of the 3D structure of crystalline albumin reveal that BSA comprises three homologous domains (I–III). Each domain is a product of subdomains that posses common structural motifs. The principal regions of binding sites are located in hydrophobic cavities in subdomains IIA and IIIA, which exhibit similar chemistry. Fig. 8 showed the best energy-ranked result of the binding mode between FNC (analogs) and BSA based on the binding-site study. The results revealed the most possible binding sites and poses for the three compounds in the protein. As shown in Fig. 8, it was obvious that the FNC molecule was surrounded by residues (LEU198, LEU203, LYS199, PHE211, TRP214, LEU481); CDPC molecule was surrounded by residues (PHE211, TRP214, LYS199, LEU198, LYS195, VAL455); while 50 -CMP was surrounded by residues (LYS195, VAL343, ALA291, PRO339, PRO447, TYR314), respectively. Meanwhile, there were also several residues in subdomains IIIA for the three systems, such as ASP451, SER454, VAL455, PRO447, SLU450. The interaction between FNC (analogs) and BSA was not exclusively hydrophobic in nature because several ionic and polar residues (ARG, ASP, GLN, SER, SLU) in the proximity of the ligand may play roles in stabilizing the molecule via hydrogen bonding and electrostatic interaction. The results of molecular modeling suggested that the interaction between FNC (analogs) and BSA was dominated by hydrophobic force, which

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