Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 133 (2014) 372–377
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Study on the interaction between carbonyl-fused N-confused porphyrin and bovine serum albumin by spectroscopic techniques Xianyong Yu a,b,c,⇑, Zhixi Liao a, Bingfei Jiang a, Lingyi Zheng a, Xiaofang Li a,⇑ a Key Laboratory of Theoretical Organic Chemistry and Function Molecule, Ministry of Education, Hunan Province College Key Laboratory of QSAR/QSPR, School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201, China b State Key Laboratory of Physical Chemistry of Solid Surfaces, Xiamen University, Xiamen 361005, China c Key Laboratory of Computational Physical Sciences, Fudan University, Ministry of Education, Shanghai, 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 interaction between BSA and CF-
300
Fluorescence intensity
NCP was studied. The fluorescence quenching mechanism is a static quenching procedure. The binding constants and binding sites were calculated. Electrostatic force played a major role in stabilizing the complex. The conformation of BSA was affected by CF-NCP.
250
a
N NH O
200
N
N
j
150 100 50 0 300
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Wavelength (nm)
a r t i c l e
i n f o
Article history: Received 22 March 2014 Received in revised form 12 May 2014 Accepted 25 May 2014 Available online 11 June 2014 Keywords: Interaction Carbonyl fused N-confused porphyrin Bovine serum albumin Fluorescence spectroscopy Ultraviolet–visible spectroscopy
a b s t r a c t The interaction between carbonyl-fused N-confused porphyrin (CF-NCP) and bovine serum albumin (BSA) was investigated by fluorescence and ultraviolet–visible (UV–Vis) spectroscopy. The results indicated that CF-NCP has strong ability to quench the intrinsic fluorescence of BSA by forming complexes. The binding constants (Ka), binding sites (n) were obtained. The corresponding thermodynamic parameters (DH, DS and DG) of the interaction system were calculated at three different temperatures. The results revealed that the binding process is spontaneous, and the acting force between CF-NCP and BSA were mainly electrostatic forces. According to Förster non-radiation energy transfer theory, the binding distance between CF-NCP and BSA was calculated to be 4.37 nm. What is more, the conformation of BSA was observed from synchronous fluorescence spectroscopy. Ó 2014 Elsevier B.V. All rights reserved.
Introduction The proteins are the most abundant macromolecules in biological cells, and meanwhile, they are also the fundamental ⇑ Corresponding authors. Address: Key Laboratory of Theoretical Organic Chemistry and Function Molecule, Ministry of Education, Hunan Province College Key Laboratory of QSAR/QSPR, School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201, China (X. Yu). Tel.: +86 731 58290187; fax: +86 731 58290509. E-mail addresses: [email protected] (X. Yu), [email protected] (X. Li). http://dx.doi.org/10.1016/j.saa.2014.05.085 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
substances that express various biological functions [1]. Serum albumins are the major soluble proteins in the circulatory system. The binding ability of drug–albumin in blood stream may have a significant impact on the distribution, free concentration, metabolism and toxicity of drugs [2,3]. Bovine serum albumin (BSA) has been one of the most extensively studied of this group of proteins, particularly because of its structural homology with human serum albumin (HSA) [4]. Besides, in this work, BSA is selected as our protein model because of its medical importance, stability, low cost, unusual ligand-binding properties [5,6].
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Porphyrins are a class of naturally occurring macrocyclic compounds used in a new technology for photodiagnosis (PD) and photodynamic therapy (PDT) of oncological and non-oncological diseases. This technology not only is effective in early tumor diagnosis but also can selectively kill the tumor organization [7,8]. N-confused porphyrin (NCP) has exhibited peculiar optical and coordination properties owing to its unique structure that possesses an NNNC core and a peripheral N atom [9] and the NCP framework can be easily modified into a novel fused porphyrinoid which called carbonyl-fused N-confused porphyrin (CF-NCP) (shown in Scheme 1) [10]. The fused ring can affect porphyrin by altering their optical properties, both ground and excited state, coordination chemistry and redox behavior, and it is this potential that has captured the attention of many scientists [11]. Some reports on the interaction between porphyrin or metalloporphyrin and serum albumin have been published, but the interaction of CF-NCP and serum albumin has seldom been reported so far [12,13]. Therefore, it is important to study the interaction between CF-NCP and BSA. Drug–protein interactions are important since most of the administered drugs are extensively and reversibly bound to serum albumin and drug is transported mainly as a complex with protein [14]. We hope this work will not only provide useful information for understanding of the CF-NCP, but also illustrate its binding mechanisms at a molecular level. Materials and methods Reagents BSA (P99%) was obtained from Huamei Bioengineering Co. (Shanghai, China) and was dissolved in a Tris–HCl (0.05 mol L1, pH = 7.43) buffer to form the BSA solution with a concentration of 1.00 105 mol L1. A Tris–HCl buffer (0.05 mol L1, pH = 7.43) containing 0.10 mol L1 NaCl was selected to keep the pH value constant and to maintain the ionic strength of the solution. CF-NCP was prepared according in the reported literature [10] and its stock solution (0.625 103 mol L1) was prepared in DMF. All other reagents were of analytical grade and doubledistilled water was used during the experiment. Apparatus Fluorescence spectra were recorded on a Shimadzu RF-5301 fluorescence spectrophotometer (Tokyo, Japan) with a SB-11 water bath (Eyela) and 1.0 cm quartz cells. The emission and excitation slits were 10 and 10 nm, respectively. The synchronous
fluorescence spectra were obtained by setting the excitation and emission wavelength interval (Dk) at 15 nm and 60 nm. The absorption spectra were obtained from a Shimadzu UV-2501 spectrophotometer (Tokyo, Japan). The pH measurement was made with a Leici pHS-2 digital pH-meter (Shanghai, China) with a combinational glass calomel electrode. Measurements of spectra A 2.5 mL solution containing 1.00 105 mol L1 BSA was titrated by successive additions of 0.625 103 mol L1 CF-NCP solution and the concentration of CO-NCP varied from 0 to 2.25 105 mol L1. Titrations were done manually by using micro-injector. Fluorescence quenching spectra were measured in the range of 280–500 nm at the excitation wavelength of 280 nm. The fluorescence spectra were performed at three temperatures (298, 304 and 310 K). The UV–Vis absorption spectra of CF-NCP solution with the concentration of 1.00 105 mol L1 was measured in the range of 200–500 nm at 292 K. Results and discussion The fluorescence quenching spectra The fluorescence spectra of BSA in the presence of CF-NCP at different concentrations are shown in Fig. 1. It is obvious that the fluorescence intensity of BSA decreased gradually with the increasing concentrations of CF-NCP and there was no significant kem shift with the addition of CF-NCP, which implying that the microenvironment around the chromophore of BSA was changed after adding CF-NCP [15]. The mechanism of quenching of BSA fluorescence by CF-NCP The fluorescence intensities were corrected for absorption of exciting light and reabsorption of emitted light using the following relationship [16,17]:
F cor ¼ F obs eðAex þAem Þ=2
ð1Þ
where Fcor and Fobs are the corrected and observed fluorescence intensities, respectively, Aex and Aem are the absorption of the system at excitation and emission wavelength, respectively. The intensity of fluorescence used in this study is always corrected. Fluorescence quenching refers to any process that decreases the fluorescence intensity of a phosphor. A variety of molecular
Fluorescence intensity
300 250
a
200 150
j
100 50
k(CF-NCP only) 0 -50 300
325
350
375
400
425
450
Wavelength (nm)
Scheme 1. Molecular structure of CF-NCP.
Fig. 1. The fluorescence quenching spectra of BSA by CF-NCP at 310 K. kex = 280 nm. [BSA] = 1.00 105 mol L1; [CF-NCP] (a–j): 0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25 (105 mol L1); [CF-NCP] (k): 2.25 105 mol L1.
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interactions can result in fluorescence quenching, including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching [18]. The different mechanisms of fluorescence quenching are usually classified as either dynamic quenching or static quenching [19]. Dynamic quenching and static quenching are caused by diffusion and ground-state complex formation, respectively. They are dependent on temperature to some extent (it is known that higher temperatures result in larger diffusion coefficients), and the dynamic quenching constants are expected to increase with increasing temperature. In contrast, increased temperature is likely to result in decreased stability of complexes, and thus result in lower values of the static quenching constants [20]. In order to clarify the quenching mechanism, the Stern–Volmer equation was used to analyze the quenching data [21]:
F0 ¼ 1 þ K sv ½Q ¼ 1 þ K q s0 ½Q F
ð2Þ
where F0 and F denote the fluorescence intensities in the absence and presence of quencher, respectively. Ksv is Stern–Volmer quenching constant; [Q] is the concentration of the quencher; Kq is the bimolecular quenching rate constant; s0 is the average lifetime of the molecule without the quencher which is about 108 s [22]. Firstly, we assumed that the procedure of the fluorescence quenching is a dynamic quenching process. The Stern–Volmer plots of the quenching of BSA fluorescence by CF-NCP at different temperatures are displayed in Fig. 2. The calculated results were shown in Table 1. For dynamic quenching, the maximum scatter collision quenching constant of various quenchers with the biopolymers is 2.0 1010 L mol1 S1 [23,24]. In addition, dynamic and static quenching can be distinguished by temperature dependence of the quenching: the values of Ksv decreased with an increasing temperature for static quenching and the reverse result will be observed for dynamic quenching. It showed that the values of Kq were greater than 2.0 1010 L mol1 S1 and the Ksv values decreased with increasing temperature, which suggested that the quenching was resulted from the formation of BSA–(CF-NCP) complex. Hence, we can say the quenching mechanism is a static quenching process [25]. UV–Vis absorption spectra UV–Vis absorption measurement is a very simple and effective method in exploring the structural change and detecting the complex formation [26]. The absorption spectra of BSA–(CF-NCP) system were measured to confirm the quenching mechanism. As
Table 1 The quenching constants of BSA by CF-NCP at different temperatures. Ksv (L mol1)
T (K) 298 304 310
Kq (L mol1 s1)
R
12
19,551 17,826 16,907
1.955 10 1.783 1012 1.691 1012
0.9954 0.9973 0.9967
shown in Fig. 3, the absorption intensity of BSA increased in the presence of CF-NCP (a) compared with the absence of CF-NCP (c), which indicated the formation of a complex [27,28]. This result reconfirmed that static quenching exists in the interaction process. Binding parameters For a static quenching interaction, the relationship between the fluorescence intensity and the concentration of quencher can be usually described by deriving to obtain the binding constant (Ka) and the number of binging sites (n) can be obtained from the following double logarithm regression curve [29,30]:
log
F0 F F0 F ¼ n log K a þ n log ½Q t n ½Bt F F0
ð3Þ
where [Qt] and [Bt] are the total concentrations of CF-NCP and BSA, respectively. The main advantage of this equation is that it uses the total concentration of quencher instead of the free concentration of quencher [31,32]. The curve of log (F0 F)/F versus log {[Qt] n[Bt](F0 F)/F0} is drawn and fitted linearly, then the value of n can be obtained from the slope of the plot. The double logarithm regression curves at different temperatures are displayed in Fig. 4. The results were shown in Table 2. The values of binding constants (Ka) decreased with the temperature increased, which may indicate that the formation of an unstable compound and the partly decomposition at high temperatures. The values of n approximate to 1, which indicated that there exists a single binding site in BSA for CF-NCP. Thermodynamic parameters and nature of the binding forces The thermodynamic parameters, such as free energy (DG), enthalpy (DH) and entropy (DS) of interaction system, are important to interpret the binding mode [33–35]. According to the theory of Ross, the positive enthalpy change DH and entropy change DS are associated with hydrophobic interaction; the negative values of DH and DS are associated with hydrogen binding and van der Waals interactions; finally, the very low
0.8 1.4 298 K 304 K 310 K
0.6
Absorbance
F0 /F
1.3 1.2
c 0.4
a
0.2
1.1 1.0
b
0.0 0.0
0.5
1.0
1.5
2.0
2.5
-5
260
280
300
320
340
360
Wavelength (nm)
[CF-NCP]/ (10 mol/L) Fig. 2. The Stern–Volmer plots of the fluorescence quenching of BSA by CF-NCP at different temperatures.
Fig. 3. UV–Vis absorption spectra of the BSA–(CF-NCP) system. [BSA] = [CFNCP] = 1.0 105 mol L1; T = 298 K; (a) BSA; (b) CF-NCP and (c) BSA + CF-NCP at the same concentration.
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Table 3 Thermodynamic parameters for BSA–(CF-NCP) system at different temperatures.
-0.4 298 K 304 K 310 K
log [(F0 -F)/F]
-0.6 -0.8 -1.0
T (K)
DH (kJ mol1)
DG (kJ mol1)
DS (J mol1 K1)
298 304 310
7.44 7.44 7.44
24.04 24.37 24.71
55.70 55.69 55.71
-1.2
Energy transfer -1.4 -1.6 -5.6
-5.4
-5.2
-5.0
-4.8
-4.6
log {[Qt ]-Bt (F0 -F)/F0 } Fig. 4. The plots of log [(F0 F)/F] versus log{Qt Bt(F0 F)/F0} at three different temperatures.
Table 2 Values of Ka, n, and R of BSA–(CF-NCP) system at different temperatures.
E¼1
T (K)
Ka (L mol1)
n
R
298 304 310
16,360 15,378 14,564
1.0114 1.0218 1.0164
0.9945 0.9990 0.9972
positive or negative DH and positive DS values are characterized by electrostatic interactions. The thermodynamic parameters of BSA–(CF-NCP) complex were calculated from the following Van’t Hoff equation:
ln K a ¼
DH 1 R T
ð4Þ
DG ¼ RT ln K a
DS ¼
DH DG T
The importance of the energy transfer in biochemistry is that the efficiency of transfer can be used to evaluate the distance between the ligand and the tryptophan residues in the protein. The overlap of the UV–Vis absorption spectra of CF-NCP with the fluorescence emission spectra of BSA is shown in Fig. 6. According to Förster theory of molecular resonance energy transfer [36,37], the distance r of binding between CF-NCP and BSA, the efficiency E of energy transfer between the donor and acceptor, could be calculated using Eq. (7):
F R6 ¼ 6 0 F0 R0 þ r 6
ð7Þ
R60 ¼ 8:8 1025 K 2 N4 uJ
ð8Þ
In Eq. (7), where E is the efficiency of transfer between the donor and the acceptor and R0 is the critical distance when the efficiency of transfer is 50%. F0 and F mean the fluorescence intensity of BSA in the absence and in the presence of quencher CF-NCP, respectively [38]. In Eq. (8), where K2 is the spatial orientation factor of the dipole, N is the refracted index of the medium, u is the fluorescence quantum yield of the donor. In this case, K2 = 2/3, N = 1.336 and u = 0.118 [39]. J can be obtained from the following equation:
ð5Þ
FðkÞeðkÞk4 Dk J¼ P FðkÞDk
ð6Þ
In Eq. (9) where J is the effect of the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, F(k) is the fluorescence intensity of the fluorescence donor at wavelength k and e(k) is the molar absorption coefficient of the acceptor at wavelength k. According to the above equations and experimental data, we obtained J = 6.53 1014 cm3 L mol1, E = 0.1681, R0 = 3.35 nm and from Eq. (7), the value of r = 4.37 nm was obtained. The distance between donor molecule and acceptor molecule is r = 4.37 (r < 7) nm, which implied a high probability of the energy transfer from BSA to CF-NCP.
where Ka stands for the binding constant at the corresponding temperature, R is the gas constant and T is the experimental temperature. The data of ln Ka versus 1/T for the (CF-NCP)–BSA system are shown in Fig. 5. The thermodynamic parameters for the interaction of CF-NCP with BSA are listed in Table 3. The negative values of DG indicated that the binding process is spontaneous, and the values of DH and DS indicated that electrostatic forces are the main force between the CF-NCP and BSA.
ð9Þ
1.0 300 lnKa = 895.37/T + 5.67
lnKa
9.68
R = 0.9998
9.64
0.8
250 200
0.6
a
150
0.4 100
b
0.2
50
9.60
0 0.00325
0.00330 -1
0.00335
-1
Absorbence
Fluorescence Intensity
9.72
0.0 300
350
400
450
500
Wavelength (nm)
T / (K ) Fig. 5. Van’t Hoff plots for the interaction of CF-NCP with BSA.
Fig. 6. The overlap of fluorescence emission spectrum of BSA (a) and absorption spectrum of CF-NCP (b); [BSA] = [CF-NCP] = 1.00 105 mol L1; T = 292 K.
X. Yu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 133 (2014) 372–377
Fluorescence intensity
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A
Fluorescence intensity
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j
30 20 10 0 280
290
300
310
320
330
a
B
300 240 180
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120 60 0 300
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340
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Fig. 7. Synchronous fluorescence spectra of BSA (A) Dk = 15 nm and (B) Dk = 60 nm. [BSA] = 1.00 105 mol L1; [CF-NCP] (a–j): 0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25 (105 mol L1).
Conformation investigation The synchronous fluorescence spectra give information about the molecular environment in a vicinity of the chromosphere molecules. It is introduced by Llody and has been used to characterize complex mixtures [40]. The spectrum characteristic of tyrosine (Tyr) residues and tryptophan (Trp) residues were observed when the wavelength interval (Dk) is 15 nm and 60 nm, respectively. Because of the red shifts of maximum emission wavelengths of both Tyr and Trp with the less hydrophobic environment, blue shifts of maximum emission wavelengths with the more hydrophobic environment occurring. These red or blue shifts indicated that the conformation of BSA has been changed [41]. It is apparent from Fig. 7 that the emission strengths of both Tyr residues and Trp residues decreased. The shift of the maximum of emission wavelength from 305 to 308 nm when Dk = 15 nm, which indicated that the conformation of BSA was changed and the polarity around the Tyr residue increased and the hydrophobicity decreased, while the Trp residues does not show significant shift [42,43]. In other words, the synchronous fluorescence spectra confirmed that the conformation of BSA has been changed. Conclusions In this paper, the interaction of BSA and CF-NCP was studied using fluorescence and absorption spectroscopy under imitated physiological conditions. The results revealed that CF-NCP could bind with BSA and the probable quenching mechanism of fluorescence of BSA by CF-NCP was a static quenching procedure. The binding constants, binding sites were also obtained. The corresponding thermodynamic parameters at different temperatures were calculated according to Van’t Hoff equation, this data indicated that the electrostatic forces played a major role in stabilizing the complex and the binding reaction was spontaneous. The synchronous fluorescence spectra showed that the structure of BSA molecules was changed in the presence of CF-NCP. The study is expected to provide important insight into the interactions of the physiologically important protein BSA with NCP derivatives and we hope the knowledge obtained in this work will be helpful to provide useful information for appropriately understanding the drug design and pharmaceutical research. Acknowledgments This work was supported by Scientific Research Fund of Hunan Provincial Education Department (12K101 and 13A026), Hunan Provincial Natural Science Foundation of China (14JJ7049), National Natural Science Foundation of China (21371054), The
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Study on the interaction between carbonyl-fused N-confused porphyrin and bovine serum albumin by spectroscopic techniques Xianyong Yu a,b,c,⇑, Zhixi Liao a, Bingfei Jiang a, Lingyi Zheng a, Xiaofang Li a,⇑ a Key Laboratory of Theoretical Organic Chemistry and Function Molecule, Ministry of Education, Hunan Province College Key Laboratory of QSAR/QSPR, School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201, China b State Key Laboratory of Physical Chemistry of Solid Surfaces, Xiamen University, Xiamen 361005, China c Key Laboratory of Computational Physical Sciences, Fudan University, Ministry of Education, Shanghai, 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 interaction between BSA and CF-
300
Fluorescence intensity
NCP was studied. The fluorescence quenching mechanism is a static quenching procedure. The binding constants and binding sites were calculated. Electrostatic force played a major role in stabilizing the complex. The conformation of BSA was affected by CF-NCP.
250
a
N NH O
200
N
N
j
150 100 50 0 300
325
350
375
400
425
450
Wavelength (nm)
a r t i c l e
i n f o
Article history: Received 22 March 2014 Received in revised form 12 May 2014 Accepted 25 May 2014 Available online 11 June 2014 Keywords: Interaction Carbonyl fused N-confused porphyrin Bovine serum albumin Fluorescence spectroscopy Ultraviolet–visible spectroscopy
a b s t r a c t The interaction between carbonyl-fused N-confused porphyrin (CF-NCP) and bovine serum albumin (BSA) was investigated by fluorescence and ultraviolet–visible (UV–Vis) spectroscopy. The results indicated that CF-NCP has strong ability to quench the intrinsic fluorescence of BSA by forming complexes. The binding constants (Ka), binding sites (n) were obtained. The corresponding thermodynamic parameters (DH, DS and DG) of the interaction system were calculated at three different temperatures. The results revealed that the binding process is spontaneous, and the acting force between CF-NCP and BSA were mainly electrostatic forces. According to Förster non-radiation energy transfer theory, the binding distance between CF-NCP and BSA was calculated to be 4.37 nm. What is more, the conformation of BSA was observed from synchronous fluorescence spectroscopy. Ó 2014 Elsevier B.V. All rights reserved.
Introduction The proteins are the most abundant macromolecules in biological cells, and meanwhile, they are also the fundamental ⇑ Corresponding authors. Address: Key Laboratory of Theoretical Organic Chemistry and Function Molecule, Ministry of Education, Hunan Province College Key Laboratory of QSAR/QSPR, School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201, China (X. Yu). Tel.: +86 731 58290187; fax: +86 731 58290509. E-mail addresses: [email protected] (X. Yu), [email protected] (X. Li). http://dx.doi.org/10.1016/j.saa.2014.05.085 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
substances that express various biological functions [1]. Serum albumins are the major soluble proteins in the circulatory system. The binding ability of drug–albumin in blood stream may have a significant impact on the distribution, free concentration, metabolism and toxicity of drugs [2,3]. Bovine serum albumin (BSA) has been one of the most extensively studied of this group of proteins, particularly because of its structural homology with human serum albumin (HSA) [4]. Besides, in this work, BSA is selected as our protein model because of its medical importance, stability, low cost, unusual ligand-binding properties [5,6].
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Porphyrins are a class of naturally occurring macrocyclic compounds used in a new technology for photodiagnosis (PD) and photodynamic therapy (PDT) of oncological and non-oncological diseases. This technology not only is effective in early tumor diagnosis but also can selectively kill the tumor organization [7,8]. N-confused porphyrin (NCP) has exhibited peculiar optical and coordination properties owing to its unique structure that possesses an NNNC core and a peripheral N atom [9] and the NCP framework can be easily modified into a novel fused porphyrinoid which called carbonyl-fused N-confused porphyrin (CF-NCP) (shown in Scheme 1) [10]. The fused ring can affect porphyrin by altering their optical properties, both ground and excited state, coordination chemistry and redox behavior, and it is this potential that has captured the attention of many scientists [11]. Some reports on the interaction between porphyrin or metalloporphyrin and serum albumin have been published, but the interaction of CF-NCP and serum albumin has seldom been reported so far [12,13]. Therefore, it is important to study the interaction between CF-NCP and BSA. Drug–protein interactions are important since most of the administered drugs are extensively and reversibly bound to serum albumin and drug is transported mainly as a complex with protein [14]. We hope this work will not only provide useful information for understanding of the CF-NCP, but also illustrate its binding mechanisms at a molecular level. Materials and methods Reagents BSA (P99%) was obtained from Huamei Bioengineering Co. (Shanghai, China) and was dissolved in a Tris–HCl (0.05 mol L1, pH = 7.43) buffer to form the BSA solution with a concentration of 1.00 105 mol L1. A Tris–HCl buffer (0.05 mol L1, pH = 7.43) containing 0.10 mol L1 NaCl was selected to keep the pH value constant and to maintain the ionic strength of the solution. CF-NCP was prepared according in the reported literature [10] and its stock solution (0.625 103 mol L1) was prepared in DMF. All other reagents were of analytical grade and doubledistilled water was used during the experiment. Apparatus Fluorescence spectra were recorded on a Shimadzu RF-5301 fluorescence spectrophotometer (Tokyo, Japan) with a SB-11 water bath (Eyela) and 1.0 cm quartz cells. The emission and excitation slits were 10 and 10 nm, respectively. The synchronous
fluorescence spectra were obtained by setting the excitation and emission wavelength interval (Dk) at 15 nm and 60 nm. The absorption spectra were obtained from a Shimadzu UV-2501 spectrophotometer (Tokyo, Japan). The pH measurement was made with a Leici pHS-2 digital pH-meter (Shanghai, China) with a combinational glass calomel electrode. Measurements of spectra A 2.5 mL solution containing 1.00 105 mol L1 BSA was titrated by successive additions of 0.625 103 mol L1 CF-NCP solution and the concentration of CO-NCP varied from 0 to 2.25 105 mol L1. Titrations were done manually by using micro-injector. Fluorescence quenching spectra were measured in the range of 280–500 nm at the excitation wavelength of 280 nm. The fluorescence spectra were performed at three temperatures (298, 304 and 310 K). The UV–Vis absorption spectra of CF-NCP solution with the concentration of 1.00 105 mol L1 was measured in the range of 200–500 nm at 292 K. Results and discussion The fluorescence quenching spectra The fluorescence spectra of BSA in the presence of CF-NCP at different concentrations are shown in Fig. 1. It is obvious that the fluorescence intensity of BSA decreased gradually with the increasing concentrations of CF-NCP and there was no significant kem shift with the addition of CF-NCP, which implying that the microenvironment around the chromophore of BSA was changed after adding CF-NCP [15]. The mechanism of quenching of BSA fluorescence by CF-NCP The fluorescence intensities were corrected for absorption of exciting light and reabsorption of emitted light using the following relationship [16,17]:
F cor ¼ F obs eðAex þAem Þ=2
ð1Þ
where Fcor and Fobs are the corrected and observed fluorescence intensities, respectively, Aex and Aem are the absorption of the system at excitation and emission wavelength, respectively. The intensity of fluorescence used in this study is always corrected. Fluorescence quenching refers to any process that decreases the fluorescence intensity of a phosphor. A variety of molecular
Fluorescence intensity
300 250
a
200 150
j
100 50
k(CF-NCP only) 0 -50 300
325
350
375
400
425
450
Wavelength (nm)
Scheme 1. Molecular structure of CF-NCP.
Fig. 1. The fluorescence quenching spectra of BSA by CF-NCP at 310 K. kex = 280 nm. [BSA] = 1.00 105 mol L1; [CF-NCP] (a–j): 0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25 (105 mol L1); [CF-NCP] (k): 2.25 105 mol L1.
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interactions can result in fluorescence quenching, including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching [18]. The different mechanisms of fluorescence quenching are usually classified as either dynamic quenching or static quenching [19]. Dynamic quenching and static quenching are caused by diffusion and ground-state complex formation, respectively. They are dependent on temperature to some extent (it is known that higher temperatures result in larger diffusion coefficients), and the dynamic quenching constants are expected to increase with increasing temperature. In contrast, increased temperature is likely to result in decreased stability of complexes, and thus result in lower values of the static quenching constants [20]. In order to clarify the quenching mechanism, the Stern–Volmer equation was used to analyze the quenching data [21]:
F0 ¼ 1 þ K sv ½Q ¼ 1 þ K q s0 ½Q F
ð2Þ
where F0 and F denote the fluorescence intensities in the absence and presence of quencher, respectively. Ksv is Stern–Volmer quenching constant; [Q] is the concentration of the quencher; Kq is the bimolecular quenching rate constant; s0 is the average lifetime of the molecule without the quencher which is about 108 s [22]. Firstly, we assumed that the procedure of the fluorescence quenching is a dynamic quenching process. The Stern–Volmer plots of the quenching of BSA fluorescence by CF-NCP at different temperatures are displayed in Fig. 2. The calculated results were shown in Table 1. For dynamic quenching, the maximum scatter collision quenching constant of various quenchers with the biopolymers is 2.0 1010 L mol1 S1 [23,24]. In addition, dynamic and static quenching can be distinguished by temperature dependence of the quenching: the values of Ksv decreased with an increasing temperature for static quenching and the reverse result will be observed for dynamic quenching. It showed that the values of Kq were greater than 2.0 1010 L mol1 S1 and the Ksv values decreased with increasing temperature, which suggested that the quenching was resulted from the formation of BSA–(CF-NCP) complex. Hence, we can say the quenching mechanism is a static quenching process [25]. UV–Vis absorption spectra UV–Vis absorption measurement is a very simple and effective method in exploring the structural change and detecting the complex formation [26]. The absorption spectra of BSA–(CF-NCP) system were measured to confirm the quenching mechanism. As
Table 1 The quenching constants of BSA by CF-NCP at different temperatures. Ksv (L mol1)
T (K) 298 304 310
Kq (L mol1 s1)
R
12
19,551 17,826 16,907
1.955 10 1.783 1012 1.691 1012
0.9954 0.9973 0.9967
shown in Fig. 3, the absorption intensity of BSA increased in the presence of CF-NCP (a) compared with the absence of CF-NCP (c), which indicated the formation of a complex [27,28]. This result reconfirmed that static quenching exists in the interaction process. Binding parameters For a static quenching interaction, the relationship between the fluorescence intensity and the concentration of quencher can be usually described by deriving to obtain the binding constant (Ka) and the number of binging sites (n) can be obtained from the following double logarithm regression curve [29,30]:
log
F0 F F0 F ¼ n log K a þ n log ½Q t n ½Bt F F0
ð3Þ
where [Qt] and [Bt] are the total concentrations of CF-NCP and BSA, respectively. The main advantage of this equation is that it uses the total concentration of quencher instead of the free concentration of quencher [31,32]. The curve of log (F0 F)/F versus log {[Qt] n[Bt](F0 F)/F0} is drawn and fitted linearly, then the value of n can be obtained from the slope of the plot. The double logarithm regression curves at different temperatures are displayed in Fig. 4. The results were shown in Table 2. The values of binding constants (Ka) decreased with the temperature increased, which may indicate that the formation of an unstable compound and the partly decomposition at high temperatures. The values of n approximate to 1, which indicated that there exists a single binding site in BSA for CF-NCP. Thermodynamic parameters and nature of the binding forces The thermodynamic parameters, such as free energy (DG), enthalpy (DH) and entropy (DS) of interaction system, are important to interpret the binding mode [33–35]. According to the theory of Ross, the positive enthalpy change DH and entropy change DS are associated with hydrophobic interaction; the negative values of DH and DS are associated with hydrogen binding and van der Waals interactions; finally, the very low
0.8 1.4 298 K 304 K 310 K
0.6
Absorbance
F0 /F
1.3 1.2
c 0.4
a
0.2
1.1 1.0
b
0.0 0.0
0.5
1.0
1.5
2.0
2.5
-5
260
280
300
320
340
360
Wavelength (nm)
[CF-NCP]/ (10 mol/L) Fig. 2. The Stern–Volmer plots of the fluorescence quenching of BSA by CF-NCP at different temperatures.
Fig. 3. UV–Vis absorption spectra of the BSA–(CF-NCP) system. [BSA] = [CFNCP] = 1.0 105 mol L1; T = 298 K; (a) BSA; (b) CF-NCP and (c) BSA + CF-NCP at the same concentration.
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Table 3 Thermodynamic parameters for BSA–(CF-NCP) system at different temperatures.
-0.4 298 K 304 K 310 K
log [(F0 -F)/F]
-0.6 -0.8 -1.0
T (K)
DH (kJ mol1)
DG (kJ mol1)
DS (J mol1 K1)
298 304 310
7.44 7.44 7.44
24.04 24.37 24.71
55.70 55.69 55.71
-1.2
Energy transfer -1.4 -1.6 -5.6
-5.4
-5.2
-5.0
-4.8
-4.6
log {[Qt ]-Bt (F0 -F)/F0 } Fig. 4. The plots of log [(F0 F)/F] versus log{Qt Bt(F0 F)/F0} at three different temperatures.
Table 2 Values of Ka, n, and R of BSA–(CF-NCP) system at different temperatures.
E¼1
T (K)
Ka (L mol1)
n
R
298 304 310
16,360 15,378 14,564
1.0114 1.0218 1.0164
0.9945 0.9990 0.9972
positive or negative DH and positive DS values are characterized by electrostatic interactions. The thermodynamic parameters of BSA–(CF-NCP) complex were calculated from the following Van’t Hoff equation:
ln K a ¼
DH 1 R T
ð4Þ
DG ¼ RT ln K a
DS ¼
DH DG T
The importance of the energy transfer in biochemistry is that the efficiency of transfer can be used to evaluate the distance between the ligand and the tryptophan residues in the protein. The overlap of the UV–Vis absorption spectra of CF-NCP with the fluorescence emission spectra of BSA is shown in Fig. 6. According to Förster theory of molecular resonance energy transfer [36,37], the distance r of binding between CF-NCP and BSA, the efficiency E of energy transfer between the donor and acceptor, could be calculated using Eq. (7):
F R6 ¼ 6 0 F0 R0 þ r 6
ð7Þ
R60 ¼ 8:8 1025 K 2 N4 uJ
ð8Þ
In Eq. (7), where E is the efficiency of transfer between the donor and the acceptor and R0 is the critical distance when the efficiency of transfer is 50%. F0 and F mean the fluorescence intensity of BSA in the absence and in the presence of quencher CF-NCP, respectively [38]. In Eq. (8), where K2 is the spatial orientation factor of the dipole, N is the refracted index of the medium, u is the fluorescence quantum yield of the donor. In this case, K2 = 2/3, N = 1.336 and u = 0.118 [39]. J can be obtained from the following equation:
ð5Þ
FðkÞeðkÞk4 Dk J¼ P FðkÞDk
ð6Þ
In Eq. (9) where J is the effect of the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, F(k) is the fluorescence intensity of the fluorescence donor at wavelength k and e(k) is the molar absorption coefficient of the acceptor at wavelength k. According to the above equations and experimental data, we obtained J = 6.53 1014 cm3 L mol1, E = 0.1681, R0 = 3.35 nm and from Eq. (7), the value of r = 4.37 nm was obtained. The distance between donor molecule and acceptor molecule is r = 4.37 (r < 7) nm, which implied a high probability of the energy transfer from BSA to CF-NCP.
where Ka stands for the binding constant at the corresponding temperature, R is the gas constant and T is the experimental temperature. The data of ln Ka versus 1/T for the (CF-NCP)–BSA system are shown in Fig. 5. The thermodynamic parameters for the interaction of CF-NCP with BSA are listed in Table 3. The negative values of DG indicated that the binding process is spontaneous, and the values of DH and DS indicated that electrostatic forces are the main force between the CF-NCP and BSA.
ð9Þ
1.0 300 lnKa = 895.37/T + 5.67
lnKa
9.68
R = 0.9998
9.64
0.8
250 200
0.6
a
150
0.4 100
b
0.2
50
9.60
0 0.00325
0.00330 -1
0.00335
-1
Absorbence
Fluorescence Intensity
9.72
0.0 300
350
400
450
500
Wavelength (nm)
T / (K ) Fig. 5. Van’t Hoff plots for the interaction of CF-NCP with BSA.
Fig. 6. The overlap of fluorescence emission spectrum of BSA (a) and absorption spectrum of CF-NCP (b); [BSA] = [CF-NCP] = 1.00 105 mol L1; T = 292 K.
X. Yu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 133 (2014) 372–377
Fluorescence intensity
60
360
a
A
Fluorescence intensity
376
50 40
j
30 20 10 0 280
290
300
310
320
330
a
B
300 240 180
j
120 60 0 300
Wavelength (nm)
320
340
360
380
Wavelength (nm)
Fig. 7. Synchronous fluorescence spectra of BSA (A) Dk = 15 nm and (B) Dk = 60 nm. [BSA] = 1.00 105 mol L1; [CF-NCP] (a–j): 0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25 (105 mol L1).
Conformation investigation The synchronous fluorescence spectra give information about the molecular environment in a vicinity of the chromosphere molecules. It is introduced by Llody and has been used to characterize complex mixtures [40]. The spectrum characteristic of tyrosine (Tyr) residues and tryptophan (Trp) residues were observed when the wavelength interval (Dk) is 15 nm and 60 nm, respectively. Because of the red shifts of maximum emission wavelengths of both Tyr and Trp with the less hydrophobic environment, blue shifts of maximum emission wavelengths with the more hydrophobic environment occurring. These red or blue shifts indicated that the conformation of BSA has been changed [41]. It is apparent from Fig. 7 that the emission strengths of both Tyr residues and Trp residues decreased. The shift of the maximum of emission wavelength from 305 to 308 nm when Dk = 15 nm, which indicated that the conformation of BSA was changed and the polarity around the Tyr residue increased and the hydrophobicity decreased, while the Trp residues does not show significant shift [42,43]. In other words, the synchronous fluorescence spectra confirmed that the conformation of BSA has been changed. Conclusions In this paper, the interaction of BSA and CF-NCP was studied using fluorescence and absorption spectroscopy under imitated physiological conditions. The results revealed that CF-NCP could bind with BSA and the probable quenching mechanism of fluorescence of BSA by CF-NCP was a static quenching procedure. The binding constants, binding sites were also obtained. The corresponding thermodynamic parameters at different temperatures were calculated according to Van’t Hoff equation, this data indicated that the electrostatic forces played a major role in stabilizing the complex and the binding reaction was spontaneous. The synchronous fluorescence spectra showed that the structure of BSA molecules was changed in the presence of CF-NCP. The study is expected to provide important insight into the interactions of the physiologically important protein BSA with NCP derivatives and we hope the knowledge obtained in this work will be helpful to provide useful information for appropriately understanding the drug design and pharmaceutical research. Acknowledgments This work was supported by Scientific Research Fund of Hunan Provincial Education Department (12K101 and 13A026), Hunan Provincial Natural Science Foundation of China (14JJ7049), National Natural Science Foundation of China (21371054), The
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