Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 231–236
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Spectroscopic studies on the interaction of Phacolysin and bovine serum albumin Xianyong Yu a,b,c,⇑, Zhixi Liao a, Qing Yao a, Heting Liu a, Wenlin Xie a,⇑ a Key Laboratory of Theoretical Chemistry and Molecular Simulation of 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
We explored the interaction of BSA
400
Fluorenscense Intensity
and PCL by spectroscopic methods. The fluorescence quenching mechanism is static quenching. The binding constants and binding sites were calculated. Hydrogen bonds and van der Waals forces were the main force in stabilizing the complex. The conformation of BSA was changed affected by PCL.
NaO 3S
H N
N
N
N H
300
SO 3Na
200
100
0 300
350
400
450
Wavelength (nm)
a r t i c l e
i n f o
Article history: Received 21 October 2013 Received in revised form 29 January 2014 Accepted 11 February 2014 Available online 27 February 2014 Keywords: Interaction Phacolysin Bovine serum albumin Spectroscopic techniques Circular dichroism spectrum
a b s t r a c t The interaction between Phacolysin (PCL) and bovine serum albumin (BSA) under imitated physiological conditions was investigated by spectroscopic (fluorescence, UV–Vis absorption and Circular dichroism) techniques. The experiments were conducted at different temperatures (294 K, 302 K, 306 K and 310 K) and the results showed that the PCL caused the fluorescence quenching of BSA through a static quenching procedure. The binding constant (Ka), binding sites (n) were obtained. The corresponding thermodynamic parameters (DH, DS and DG) of the interaction system were calculated at different temperatures. The results revealed that the binding process was spontaneous and the acting force between PCL and BSA were mainly hydrogen bonding and van der Waals forces. According to Förster non-radiation energy transfer theory, the binding distance between PCL and BSA was calculated to be 2.41 nm. What is more, both synchronous fluorescence and Circular dichroism spectra confirmed the interaction, which indicated the conformational changes of BSA. Ó 2014 Elsevier B.V. All rights reserved.
Introduction ⇑ Corresponding authors. Address: Key Laboratory of Theoretical Chemistry and Molecular Simulation of 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] (W. Xie). http://dx.doi.org/10.1016/j.saa.2014.02.064 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
As we all know, serum albumins (SA) contribute to colloid osmotic blood pressure and the maintenance of blood pH. They also play a dominant role in drug disposition and efficacy. Most drugs are transported as a complex with SA, which makes SA an important part of drug metabolism [1–3]. The serum protein used
X. Yu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 231–236
NaO3S
H N
N
N
N H
SO3Na
Scheme 1. Molecular structure of Phacolysin.
in these studies is bovine serum albumin (BSA) which has an inherent fluorescing property attributed to the presence of aromatic amino acids [4–6]. BSA is a natural globular protein with a relatively high molecular mass (68,000 Da) and a single polypeptide chain containing 583 amino acid residues. It consists of three linearly arranged and structurally distinct homologous domains (I–III), which were divided into nine loops (L1–L9). Each domain contains two sub-domains (A and B). What’s more, tertiary structures of BSA and human serum albumin (HSA) are similar in 76% and all the study results are consistent with the fact that HSA and BSA are homologous protein [7,8]. Phacolysin (PCL) (Scheme 1) also known as Phacolin, which is a proteolytic enzyme activator. As eye drops, it can prevent cataracts [9,10]. When instill it into the denatured protein and once it is absorbed, it can promote the degradation of protein and penetrate into the lens. Thus it can maintain the transparency of the lens, improve eye tissues metabolism and prevent from the progression of cataract [11,12]. Protein–drug interaction is the hot point in the fields of medicine, chemistry and biology [13]. Many drugs and other bioactive small molecules bind reversibly to albumin, which then function as carriers [14]. Consequently, it is important to study the interactions of drugs with this protein. In this paper, the binding of Phacolysin to BSA was studied under imitated physiological conditions by fluorescence, ultraviolet spectroscopy and Circular dichroism spectrum. The binding constants were calculated and binding mechanism was investigated. In addition, the effect of PCL on the conformational change of BSA was also studied. We hope this work will not only provide useful information for understanding of the PCL, 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. Phacolysin was obtained from Liye Pharmaceutical Co. (Nanjing, China). The Phacolysin (6.14 105 mol L1) solution was prepared in double–distilled water. All other reagents were of analytical grade and double–distilled 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 nm 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). Circular dichroism spectra were recorded on Chirascan (London, UK). 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 0.614 106 mol L1 PCL solution and the concentration of PCL varied from 0 to 5.53 106 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 (294, 302 and 310 K). The UV–Vis absorption spectra of PCL solution with the concentration of 1.00 105 mol L1 was measured in the range of 240–500 nm at 294 K. The CD measurements of BSA in the presence and absence of PCL were made in the range of 195–260 nm at 293 K. A stock solution of 0.20 lmol L1 BSA was prepared in Tris–HCl buffer (0.05 mol L1) of pH 7.43 containing 0.10 mol L1 NaCl. The molar ratios of PCL to BSA were varied from 0, 1:1 to 5:1 and the CD spectrum was recorded. Results and discussion Calculating method Absorption of Phacolysin at the emission and excitation wavelength of fluorophore has an important effect on the fluorescence spectra, so the fluorescence intensity must be corrected. When the absorbance of drugs was lower than 0.3, the following equation can be used to correct the inner filter effects [15–17]:
F corr ¼ F obs eðAex þAem Þ=2
ð1Þ
where Fcorr is the corrected fluorescence intensity, Fobs is the observed intensity. Aem and Aex are the absorbance values of PCL at emission and excitation wavelengths, respectively. The fluorescence intensity used in this paper was corrected. The fluorescence quenching spectra Fig. 1 shows the emission spectra of BSA in the presence of various concentrations of PCL. It was observed that the fluorescence intensity of BSA decreased with the increasing concentration of
350 300
Fluorenscense Intensity
232
a
250 200
j
150 100 50 0 300
325
350
375
400
425
450
Wavelength (nm) Fig. 1. The fluorescence quenching spectra of BSA by PCL at 310 K. kex = 280 nm. [BSA] = 1.00 105 mol L1; [PCL] (a–j): 0, 0.614, 1.23, 1.84, 2.46, 3.07, 3.68, 4.30, 4.91, 5.53 (106 mol L1).
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PCL. It is obvious that BSA has a strong fluorescence emission peak at 342 nm when excited the wavelength at 280 nm, the fluorescence intensity of BSA decreased regularly with the addition of PCL. But there was no significant kem shift with the addition of PCL when a fixed concentration of BSA was titrated with increasing concentration of PCL. These data indicated that PCL can interact with BSA and quench its fluorescence.
Table 1 The quenching constants of BSA by PCL at different temperatures. Ksv (L mol1)
T (K)
4
294 302 306 310 a
5.289 10 4.715 104 4.414 104 3.728 104
Kq (L mol1 s1) 12
5.289 10 4.715 1012 4.414 1012 3.728 1012
R
RSDa (%)
0.9949 0.9978 0.9942 0.9878
0.0000605 0.0000339 0.0000770 0.0000156
RSD is relative standard deviation for Ksv.
The fluorescence quenching mechanism Fluorescence quenching refers to any process that decreases the fluorescence intensity of a sample. A variety of molecular interactions can result in quenching, including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching [18]. Fluorescence quenching can be dynamic, resulting from collisional encounters between the fluorophore and quencher, or static, resulting from the formation of a ground state complex between the fluorophore and quencher. Static and dynamic quenching can be distinguished by their different dependence on temperature and viscosity, or excited-state lifetime. Higher temperatures will result in faster diffusion and hence larger amounts of collision quenching, which results in the dissociation of weakly bound complexes and hence smaller amounts of static quenching. The possible quenching mechanism can be interpreted by the Stern–Volmer equation [19,20]:
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. Kq is the bimolecular quenching rate constant; s0 is the average lifetime of the molecule without the quencher which is about 108 s. Ksv is Stern–Volmer quenching constant; [Q] is the concentration of the quencher [21,22]. The values of Ksv decreased with an increasing temperature for static quenching and the reverse result will be observed for dynamic quenching. In order to confirm the mechanism of the fluorescence quenching, we firstly assumed that the procedure of the fluorescence quenching is a dynamic quenching process. The Stern–Volmer plots of the quenching of BSA fluorescence by PCL 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. 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–PCL complex. Hence, we can say the quenching mechanism is a static quenching process [23]. 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 [24,25]. For confirming the static quenching effect may exist in the system of PCL and BSA, the absorption spectra of BSA in the presence PCL were recorded and presented in Fig. 3. As can be seen from Fig. 3, with the addition of PCL (curves a–j) the absorbance intensity increased obviously and the absorption spectra maximum shifted towards shorter wavelength region, which indicate that BSA can bind to PCL and form the BSA–PCL complex. This result reconfirmed that static quenching exists in the interaction process. Binding parameters The binding constant (Ka) and the number of binging sites (n) can be obtained from the following double logarithm regression curve [26–29]:
log
F0 F F0 F ¼ n log K a þ n log ½Q t n ½Bt F F0
ð3Þ
where F0 and F are the fluorescence intensities before and after the addition of the quencher, [Qt] and [Bt] are the total concentrations of PCL 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. Ka and n values can be obtained by the plot of log (F0 F)/F vs. log {[Qt] [Bt](F0 F)/F0}. The double
1.4 0.75
294 K 302 K 306 K 310 K
1.2
0.6
a
0.0 240
3
[ Phacolysin]/(10-6
4
5
6
0.55
λ= 280 nm
0.45
a
1.0 2
0.60
0.50
1.1
1
0.70 0.65
0.3
0
j Absorbance
1.3
Absorbance
Flourescence Intensity
0.9
0
1
2
3
4
5
6
[PCL]/(10-6mol/L)
260
280
300
320
340
Wavelength (nm)
mol/L)
Fig. 2. The Stern–Volmer plots of the fluorescence quenching of BSA by PCL at different temperatures.
Fig. 3. Absorbance spectra of BSA in the presence of various PCL concentrations. [BSA] = 1.00 105 mol L1; [PCL] (a–j): 0, 0.614, 1.23, 1.84, 2.46, 3.07, 3.68, 4.3, 4.91, 5.53 (106 mol L1).
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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 roughly equal to 1, which indicated the existence of just one binding site in BSA for PCL.
Table 3 Thermodynamic parameters for BSA–PCL system at different temperatures.
Determination of the acting force
temperature. The values of DG, DH, and DS are listed in Table 3. The negative values of DG indicates that the binding process is spontaneous, while the negative values of DH and DS indicated that the forces between the PCL and BSA molecules were mainly hydrogen bonds and van der Waals interactions.
The thermodynamic parameters, such as free energy (DG), enthalpy (DH) and entropy (DS) of interaction system, are important to interpret the binding mode [30–32]. 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 positive or negative DH and positive DS values are characterized by electrostatic interactions. If there is no significant change in temperature, DH can be regarded as a constant and calculated by linear fitting plot of ln Ka vs. 1/T based on the following Eq. (4). Then DG can be obtained from Eq. (5), and DS can be obtained from Eq. (6):
ln K a ¼
DH 1 R T
ð4Þ
DG ¼ RT ln K a
T (K)
DH (kJ mol1)
DG (kJ mol1)
DS (J mol1 K1)
294 302 306 310
28.95 28.95 28.95 28.95
28.37 28.77 28.83 28.22
1.973 0.596 0.392 2.355
Energy transfer 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 [33]. The overlap of the UV–Vis absorption spectra of PCL with the fluorescence emission spectra of BSA is shown in Fig. 5. According to Förster theory of molecular resonance energy transfer [34], the distance r of binding between PCL and BSA, the efficiency E of energy transfer between the donor and acceptor, could be calculated using Eq. (7):
ð5Þ E¼1
DH DG DS ¼ T
ð6Þ
where Ka stands for the binding constant at the corresponding temperature, R is the gas constant and T is the experimental
-0.4 294 K 302 K 306 K 310 K
-1.2
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 (PCL), respectively [35]. 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 [36]. J can be obtained from the following equation:
J¼
log
[(F0 -F)/F]
-0.8
F R6 ¼ 6 0 F0 R0 þ r6
FðkÞeðkÞk4 Dk P FðkÞDk
ð9Þ
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
-1.6
1.0 -6.2
-6.0
-5.8
-5.6
log { [Qt ]-Bt (F0 -F)/F0 }
Table 2 Values of Ka, n, and R of BSA–PCL system at different temperatures.
c
T (K)
Ka (105 L mol1)
RSDb (%)
n
RSDc (%)
R
294 302 306 310
1.099 0.9484 0.8369 0.5689
0.00000819 0.00000358 0.00000442 0.00000689
1.2062 1.2102 1.2091 1.0998
0.75 0.33 0.46 0.53
0.9967 0.9969 0.9944 0.9794
RSD is relative standard deviation for Ka, RSD is relative standard deviation for n.
0.8 240 0.6
a
160
0.4
80
0.2
b 0
300
Absorbencet
Fig. 4. The plots of log [(F0 F)/F] vs. log {Qt Bt(F0 F)/F0} at four different temperatures.
b
320
-5.4
Fluorenscense Intensity
-6.4
350
400
0.0 450
Wavelength (nm) Fig. 5. The overlap of fluorescence emission spectrum of BSA (a) and absorption spectrum of PCL (b); [BSA] = [PCL] = 1.00 105 mol L1; T = 310 K.
235
60
a
A
Fluorenscense Intensity
Fluorenscense Intensity
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50 40 j
30 20 10 0 280
290
300
310
320
330
340
400
a
B
300 200
j
100 0
310
320
Wavelength (nm)
330
340
350
360
370
Wavelength (nm)
Fig. 6. Synchronous fluorescence spectra of BSA. (A) Dk ¼ 15 nm; (B) Dk ¼ 60 nm. [BSA] = 1.00 105 mol L1; [PCL] (a–j): 0, 0.614, 1.23, 1.84, 2.46, 3.07, 3.68, 4.30, 4.91, 5.53 (106 mol L1).
Conformation investigation Synchronous fluorescence spectrum 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 [38]. The spectrum characteristic of tyrosine residues and tryptophan residues were observed when the wavelength interval (Dk) is 15 nm and 60 nm, respectively [39]. The shifts of the maximum emission wavelength of the residues correspond to the polarity around the chromophore molecule, so we can evaluate the conformation changes of BSA by the shift in position of the emission maximum [40]. It is apparent from Fig. 6 that the emission strengths of both tyrosine residues and tryptophan residues decreased. The maximum of emission showed a visible red shift when Dk = 15 nm, which indicated that the conformation of BSA was changed and the polarity around the tyrosine residue increased and the hydrophobicity decreased, while when Dk = 60 nm, the tryptophan residues does not show significant shift [41,42]. In other words, the synchronous fluorescence spectra confirmed that the conformation of BSA has been changed. CD spectrum CD is widely used for the determination of the secondary structure of macromolecules [43]. Fig. 7 showed the CD spectra of BSA in the absence and presence of PCL at 293 K. In this work, the molar ratios of 0:1, 1:1, and 5:1 for PCL: BSA was used for the CD measurements. The CD spectra of BSA exhibited two negative bands in the UV region at 208 and 222 nm, characteristic of an a-helical structure of protein [44]. The CD results were expressed in terms of mean residue ellipticity (MRE) in deg cm2 dmol1 according to the following equation [24]:
Observed CDðm degÞ MRE ¼ C p n l 10
ð10Þ
where Cp is the molar concentration of the protein, n is the number of amino acid residues and l is the path length. The a-helical contents of free and combined BSA were calculated from MRE values at 208 nm using the equation [45]:
-5
-10
2
-1
θ ( deg· cm · dmol )
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 = 4.432 1015 cm3 L mol1, E = 0.3284, R0 = 2.14 nm. From the Eq. (7), the value of r = 2.41 nm was obtained. The distance between donor molecule and acceptor molecule is r = 2.41 (r < 7) nm, which indicated that the non-radiative energy transfer from PCL to BSA [8,37].
-15
c -20
a -25 200
210
220
230
240
Wavelength (nm) Fig. 7. Circular dichroism (CD) spectrum of BSA with different molar ratios of PCL to BSA, (a–c): 0, 1:1, 5:1; [BSA]=0.20 lmol L1; T = 293 K.
a-helixð%Þ ¼
MRE208 4000 100 33; 000 4000
ð11Þ
where MRE208 is the observed MRE value at 208 nm, 4000 is the MRE of the b-form and random coil conformation cross at 208 nm and 33000 is the MRE value of a pure a-helix at 208 nm. From Eqs. (10) and (11), the a-helicity in the secondary structure of BSA can be determined. In this experiment, Cp = 0.20 lmol L1, n = 583, l = 1.0 cm. According to the above equations and experimental data, the values of a-helix (%) were 56.50% in free BSA, 54.90% in the PCL-BSA complex (molar ratio of PCL to BSA 1:1) and 50.50% with molar ratio 5:1, respectively. The data were indicative of the loss of a-helix due to the interaction of PCL with BSA. The percentage of protein ahelix structure decreased indicated that PCL bound with the amino acid residue of the main polypeptide chain of BSA and destroyed their hydrogen bonding networks, which indicated that PCL has changed the secondary structure of BSA [46]. Conclusions This paper investigated the interaction between BSA and PCL using fluorescence, UV–Vis and CD techniques under imitated physiological conditions. The results revealed that PCL could bind with BSA and the probable quenching mechanism of fluorescence of BSA by PCL was static. The binding constants, binding sites were obtained. What is more, the corresponding thermodynamic parameters at different temperatures were calculated according to Van’t Hoff equation, these data indicated that the hydrogen binds and
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van der Waals force played a major role in stabilizing the complex and the binding reaction was spontaneous. The synchronous fluorescence spectra and Circular dichroism spectra showed that the structure of BSA was changed in the presence of PCL. Acknowledgments This work was supported by Scientific Research Fund of Hunan Provincial Education Department (12K101), Hunan Provincial Natural Science Foundation of China (14JJ7049), the Scientific Research Fund of Hunan Provincial Education Department (No. 13A028) and Science and Technology Project of Hunan Province (No. 2013SK3161), the Opening Project of State Key Laboratory of Physical Chemistry of Solid Surfaces (Xiamen University, No. 201309), Aid program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province. References [1] B. Liu, Y. Guo, J. Wang, R. Xu, X. Wang, D. Wang, L.Q. Zhang, Y.N. Xu, J. Lumin. 130 (2010) 1036–1043. [2] Y.J. Hu, Y. Liu, L.X. Zhang, R.M. Zhao, S.S. Qu, J. Mol. Struct. 750 (2005) 174–178. [3] A. Sułkowska, J. Rownicka, B. Bojko, J. Pozycka, I. Zubik-Skupien, W. Sułkowski, J. Mol. Struct. 704 (2004) 291–295. [4] J.Q. Gao, Y.W. Guo, J. Wang, Z.Q. Wang, Spectrochim. Acta A 78 (2011) 1278– 1286. [5] A. Kathiravan, M. Chandramohan, R. Renganathan, S. Sekar, J. Mol. Struct. 919 (2009) 210–214. [6] D. Mallik, A. Mir, S. Bhattacharya, S. Nayar, Spectrochim. Acta A 78 (2011) 332– 336. [7] J.N. Tian, J.Q. Liu, X. Tian, Z.D. Hu, X.G. Chen, J. Mol. Struct. 691 (2004) 197–202. [8] S.Y. Bi, D.Q. Song, Y.H. Kan, D. Xu, Y. Tian, X. Zhou, H.Q. Zhang, Spectrochim. Acta A 62 (2005) 203–212. [9] G. Brian, H. Taylor, Bull. World Health Organ. 79 (2001) 249–256. [10] E.M. Genies, M. Lapkowski, J.F. PenneauNew, J. Electroanal. Chem. 249 (1988) 97–107. [11] W. Wu, X.L. Tang, M.F. He, M.L. Chen, J. Chin. Pharm. 15 (2004) 48–49. [12] G.F. Feng, Y.Y. Wang, H.H. Li, Y.P. Zhao, Y.P. Xiao, J. Fine Chem. Intermed. 42 (2012) 32–35. [13] H.D. Yuan, J. Pawliszyn, Anal. Chem. 73 (2001) 4410–4416. [14] Y.J. Hu, Y. Liu, W. Jiang, R.M. Zhao, S.S. Qu, J. Photochem. Photobiol., B 80 (2005) 235–242. [15] R.F. Steiner, I. Weinryb, Excited States of Protein and Nucleic Acid, Plenum Press, New York, 1971. pp. 39–40.
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Spectroscopic studies on the interaction of Phacolysin and bovine serum albumin Xianyong Yu a,b,c,⇑, Zhixi Liao a, Qing Yao a, Heting Liu a, Wenlin Xie a,⇑ a Key Laboratory of Theoretical Chemistry and Molecular Simulation of 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
We explored the interaction of BSA
400
Fluorenscense Intensity
and PCL by spectroscopic methods. The fluorescence quenching mechanism is static quenching. The binding constants and binding sites were calculated. Hydrogen bonds and van der Waals forces were the main force in stabilizing the complex. The conformation of BSA was changed affected by PCL.
NaO 3S
H N
N
N
N H
300
SO 3Na
200
100
0 300
350
400
450
Wavelength (nm)
a r t i c l e
i n f o
Article history: Received 21 October 2013 Received in revised form 29 January 2014 Accepted 11 February 2014 Available online 27 February 2014 Keywords: Interaction Phacolysin Bovine serum albumin Spectroscopic techniques Circular dichroism spectrum
a b s t r a c t The interaction between Phacolysin (PCL) and bovine serum albumin (BSA) under imitated physiological conditions was investigated by spectroscopic (fluorescence, UV–Vis absorption and Circular dichroism) techniques. The experiments were conducted at different temperatures (294 K, 302 K, 306 K and 310 K) and the results showed that the PCL caused the fluorescence quenching of BSA through a static quenching procedure. The binding constant (Ka), binding sites (n) were obtained. The corresponding thermodynamic parameters (DH, DS and DG) of the interaction system were calculated at different temperatures. The results revealed that the binding process was spontaneous and the acting force between PCL and BSA were mainly hydrogen bonding and van der Waals forces. According to Förster non-radiation energy transfer theory, the binding distance between PCL and BSA was calculated to be 2.41 nm. What is more, both synchronous fluorescence and Circular dichroism spectra confirmed the interaction, which indicated the conformational changes of BSA. Ó 2014 Elsevier B.V. All rights reserved.
Introduction ⇑ Corresponding authors. Address: Key Laboratory of Theoretical Chemistry and Molecular Simulation of 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] (W. Xie). http://dx.doi.org/10.1016/j.saa.2014.02.064 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
As we all know, serum albumins (SA) contribute to colloid osmotic blood pressure and the maintenance of blood pH. They also play a dominant role in drug disposition and efficacy. Most drugs are transported as a complex with SA, which makes SA an important part of drug metabolism [1–3]. The serum protein used
X. Yu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 231–236
NaO3S
H N
N
N
N H
SO3Na
Scheme 1. Molecular structure of Phacolysin.
in these studies is bovine serum albumin (BSA) which has an inherent fluorescing property attributed to the presence of aromatic amino acids [4–6]. BSA is a natural globular protein with a relatively high molecular mass (68,000 Da) and a single polypeptide chain containing 583 amino acid residues. It consists of three linearly arranged and structurally distinct homologous domains (I–III), which were divided into nine loops (L1–L9). Each domain contains two sub-domains (A and B). What’s more, tertiary structures of BSA and human serum albumin (HSA) are similar in 76% and all the study results are consistent with the fact that HSA and BSA are homologous protein [7,8]. Phacolysin (PCL) (Scheme 1) also known as Phacolin, which is a proteolytic enzyme activator. As eye drops, it can prevent cataracts [9,10]. When instill it into the denatured protein and once it is absorbed, it can promote the degradation of protein and penetrate into the lens. Thus it can maintain the transparency of the lens, improve eye tissues metabolism and prevent from the progression of cataract [11,12]. Protein–drug interaction is the hot point in the fields of medicine, chemistry and biology [13]. Many drugs and other bioactive small molecules bind reversibly to albumin, which then function as carriers [14]. Consequently, it is important to study the interactions of drugs with this protein. In this paper, the binding of Phacolysin to BSA was studied under imitated physiological conditions by fluorescence, ultraviolet spectroscopy and Circular dichroism spectrum. The binding constants were calculated and binding mechanism was investigated. In addition, the effect of PCL on the conformational change of BSA was also studied. We hope this work will not only provide useful information for understanding of the PCL, 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. Phacolysin was obtained from Liye Pharmaceutical Co. (Nanjing, China). The Phacolysin (6.14 105 mol L1) solution was prepared in double–distilled water. All other reagents were of analytical grade and double–distilled 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 nm 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). Circular dichroism spectra were recorded on Chirascan (London, UK). 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 0.614 106 mol L1 PCL solution and the concentration of PCL varied from 0 to 5.53 106 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 (294, 302 and 310 K). The UV–Vis absorption spectra of PCL solution with the concentration of 1.00 105 mol L1 was measured in the range of 240–500 nm at 294 K. The CD measurements of BSA in the presence and absence of PCL were made in the range of 195–260 nm at 293 K. A stock solution of 0.20 lmol L1 BSA was prepared in Tris–HCl buffer (0.05 mol L1) of pH 7.43 containing 0.10 mol L1 NaCl. The molar ratios of PCL to BSA were varied from 0, 1:1 to 5:1 and the CD spectrum was recorded. Results and discussion Calculating method Absorption of Phacolysin at the emission and excitation wavelength of fluorophore has an important effect on the fluorescence spectra, so the fluorescence intensity must be corrected. When the absorbance of drugs was lower than 0.3, the following equation can be used to correct the inner filter effects [15–17]:
F corr ¼ F obs eðAex þAem Þ=2
ð1Þ
where Fcorr is the corrected fluorescence intensity, Fobs is the observed intensity. Aem and Aex are the absorbance values of PCL at emission and excitation wavelengths, respectively. The fluorescence intensity used in this paper was corrected. The fluorescence quenching spectra Fig. 1 shows the emission spectra of BSA in the presence of various concentrations of PCL. It was observed that the fluorescence intensity of BSA decreased with the increasing concentration of
350 300
Fluorenscense Intensity
232
a
250 200
j
150 100 50 0 300
325
350
375
400
425
450
Wavelength (nm) Fig. 1. The fluorescence quenching spectra of BSA by PCL at 310 K. kex = 280 nm. [BSA] = 1.00 105 mol L1; [PCL] (a–j): 0, 0.614, 1.23, 1.84, 2.46, 3.07, 3.68, 4.30, 4.91, 5.53 (106 mol L1).
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PCL. It is obvious that BSA has a strong fluorescence emission peak at 342 nm when excited the wavelength at 280 nm, the fluorescence intensity of BSA decreased regularly with the addition of PCL. But there was no significant kem shift with the addition of PCL when a fixed concentration of BSA was titrated with increasing concentration of PCL. These data indicated that PCL can interact with BSA and quench its fluorescence.
Table 1 The quenching constants of BSA by PCL at different temperatures. Ksv (L mol1)
T (K)
4
294 302 306 310 a
5.289 10 4.715 104 4.414 104 3.728 104
Kq (L mol1 s1) 12
5.289 10 4.715 1012 4.414 1012 3.728 1012
R
RSDa (%)
0.9949 0.9978 0.9942 0.9878
0.0000605 0.0000339 0.0000770 0.0000156
RSD is relative standard deviation for Ksv.
The fluorescence quenching mechanism Fluorescence quenching refers to any process that decreases the fluorescence intensity of a sample. A variety of molecular interactions can result in quenching, including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching [18]. Fluorescence quenching can be dynamic, resulting from collisional encounters between the fluorophore and quencher, or static, resulting from the formation of a ground state complex between the fluorophore and quencher. Static and dynamic quenching can be distinguished by their different dependence on temperature and viscosity, or excited-state lifetime. Higher temperatures will result in faster diffusion and hence larger amounts of collision quenching, which results in the dissociation of weakly bound complexes and hence smaller amounts of static quenching. The possible quenching mechanism can be interpreted by the Stern–Volmer equation [19,20]:
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. Kq is the bimolecular quenching rate constant; s0 is the average lifetime of the molecule without the quencher which is about 108 s. Ksv is Stern–Volmer quenching constant; [Q] is the concentration of the quencher [21,22]. The values of Ksv decreased with an increasing temperature for static quenching and the reverse result will be observed for dynamic quenching. In order to confirm the mechanism of the fluorescence quenching, we firstly assumed that the procedure of the fluorescence quenching is a dynamic quenching process. The Stern–Volmer plots of the quenching of BSA fluorescence by PCL 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. 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–PCL complex. Hence, we can say the quenching mechanism is a static quenching process [23]. 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 [24,25]. For confirming the static quenching effect may exist in the system of PCL and BSA, the absorption spectra of BSA in the presence PCL were recorded and presented in Fig. 3. As can be seen from Fig. 3, with the addition of PCL (curves a–j) the absorbance intensity increased obviously and the absorption spectra maximum shifted towards shorter wavelength region, which indicate that BSA can bind to PCL and form the BSA–PCL complex. This result reconfirmed that static quenching exists in the interaction process. Binding parameters The binding constant (Ka) and the number of binging sites (n) can be obtained from the following double logarithm regression curve [26–29]:
log
F0 F F0 F ¼ n log K a þ n log ½Q t n ½Bt F F0
ð3Þ
where F0 and F are the fluorescence intensities before and after the addition of the quencher, [Qt] and [Bt] are the total concentrations of PCL 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. Ka and n values can be obtained by the plot of log (F0 F)/F vs. log {[Qt] [Bt](F0 F)/F0}. The double
1.4 0.75
294 K 302 K 306 K 310 K
1.2
0.6
a
0.0 240
3
[ Phacolysin]/(10-6
4
5
6
0.55
λ= 280 nm
0.45
a
1.0 2
0.60
0.50
1.1
1
0.70 0.65
0.3
0
j Absorbance
1.3
Absorbance
Flourescence Intensity
0.9
0
1
2
3
4
5
6
[PCL]/(10-6mol/L)
260
280
300
320
340
Wavelength (nm)
mol/L)
Fig. 2. The Stern–Volmer plots of the fluorescence quenching of BSA by PCL at different temperatures.
Fig. 3. Absorbance spectra of BSA in the presence of various PCL concentrations. [BSA] = 1.00 105 mol L1; [PCL] (a–j): 0, 0.614, 1.23, 1.84, 2.46, 3.07, 3.68, 4.3, 4.91, 5.53 (106 mol L1).
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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 roughly equal to 1, which indicated the existence of just one binding site in BSA for PCL.
Table 3 Thermodynamic parameters for BSA–PCL system at different temperatures.
Determination of the acting force
temperature. The values of DG, DH, and DS are listed in Table 3. The negative values of DG indicates that the binding process is spontaneous, while the negative values of DH and DS indicated that the forces between the PCL and BSA molecules were mainly hydrogen bonds and van der Waals interactions.
The thermodynamic parameters, such as free energy (DG), enthalpy (DH) and entropy (DS) of interaction system, are important to interpret the binding mode [30–32]. 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 positive or negative DH and positive DS values are characterized by electrostatic interactions. If there is no significant change in temperature, DH can be regarded as a constant and calculated by linear fitting plot of ln Ka vs. 1/T based on the following Eq. (4). Then DG can be obtained from Eq. (5), and DS can be obtained from Eq. (6):
ln K a ¼
DH 1 R T
ð4Þ
DG ¼ RT ln K a
T (K)
DH (kJ mol1)
DG (kJ mol1)
DS (J mol1 K1)
294 302 306 310
28.95 28.95 28.95 28.95
28.37 28.77 28.83 28.22
1.973 0.596 0.392 2.355
Energy transfer 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 [33]. The overlap of the UV–Vis absorption spectra of PCL with the fluorescence emission spectra of BSA is shown in Fig. 5. According to Förster theory of molecular resonance energy transfer [34], the distance r of binding between PCL and BSA, the efficiency E of energy transfer between the donor and acceptor, could be calculated using Eq. (7):
ð5Þ E¼1
DH DG DS ¼ T
ð6Þ
where Ka stands for the binding constant at the corresponding temperature, R is the gas constant and T is the experimental
-0.4 294 K 302 K 306 K 310 K
-1.2
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 (PCL), respectively [35]. 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 [36]. J can be obtained from the following equation:
J¼
log
[(F0 -F)/F]
-0.8
F R6 ¼ 6 0 F0 R0 þ r6
FðkÞeðkÞk4 Dk P FðkÞDk
ð9Þ
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
-1.6
1.0 -6.2
-6.0
-5.8
-5.6
log { [Qt ]-Bt (F0 -F)/F0 }
Table 2 Values of Ka, n, and R of BSA–PCL system at different temperatures.
c
T (K)
Ka (105 L mol1)
RSDb (%)
n
RSDc (%)
R
294 302 306 310
1.099 0.9484 0.8369 0.5689
0.00000819 0.00000358 0.00000442 0.00000689
1.2062 1.2102 1.2091 1.0998
0.75 0.33 0.46 0.53
0.9967 0.9969 0.9944 0.9794
RSD is relative standard deviation for Ka, RSD is relative standard deviation for n.
0.8 240 0.6
a
160
0.4
80
0.2
b 0
300
Absorbencet
Fig. 4. The plots of log [(F0 F)/F] vs. log {Qt Bt(F0 F)/F0} at four different temperatures.
b
320
-5.4
Fluorenscense Intensity
-6.4
350
400
0.0 450
Wavelength (nm) Fig. 5. The overlap of fluorescence emission spectrum of BSA (a) and absorption spectrum of PCL (b); [BSA] = [PCL] = 1.00 105 mol L1; T = 310 K.
235
60
a
A
Fluorenscense Intensity
Fluorenscense Intensity
X. Yu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 231–236
50 40 j
30 20 10 0 280
290
300
310
320
330
340
400
a
B
300 200
j
100 0
310
320
Wavelength (nm)
330
340
350
360
370
Wavelength (nm)
Fig. 6. Synchronous fluorescence spectra of BSA. (A) Dk ¼ 15 nm; (B) Dk ¼ 60 nm. [BSA] = 1.00 105 mol L1; [PCL] (a–j): 0, 0.614, 1.23, 1.84, 2.46, 3.07, 3.68, 4.30, 4.91, 5.53 (106 mol L1).
Conformation investigation Synchronous fluorescence spectrum 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 [38]. The spectrum characteristic of tyrosine residues and tryptophan residues were observed when the wavelength interval (Dk) is 15 nm and 60 nm, respectively [39]. The shifts of the maximum emission wavelength of the residues correspond to the polarity around the chromophore molecule, so we can evaluate the conformation changes of BSA by the shift in position of the emission maximum [40]. It is apparent from Fig. 6 that the emission strengths of both tyrosine residues and tryptophan residues decreased. The maximum of emission showed a visible red shift when Dk = 15 nm, which indicated that the conformation of BSA was changed and the polarity around the tyrosine residue increased and the hydrophobicity decreased, while when Dk = 60 nm, the tryptophan residues does not show significant shift [41,42]. In other words, the synchronous fluorescence spectra confirmed that the conformation of BSA has been changed. CD spectrum CD is widely used for the determination of the secondary structure of macromolecules [43]. Fig. 7 showed the CD spectra of BSA in the absence and presence of PCL at 293 K. In this work, the molar ratios of 0:1, 1:1, and 5:1 for PCL: BSA was used for the CD measurements. The CD spectra of BSA exhibited two negative bands in the UV region at 208 and 222 nm, characteristic of an a-helical structure of protein [44]. The CD results were expressed in terms of mean residue ellipticity (MRE) in deg cm2 dmol1 according to the following equation [24]:
Observed CDðm degÞ MRE ¼ C p n l 10
ð10Þ
where Cp is the molar concentration of the protein, n is the number of amino acid residues and l is the path length. The a-helical contents of free and combined BSA were calculated from MRE values at 208 nm using the equation [45]:
-5
-10
2
-1
θ ( deg· cm · dmol )
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 = 4.432 1015 cm3 L mol1, E = 0.3284, R0 = 2.14 nm. From the Eq. (7), the value of r = 2.41 nm was obtained. The distance between donor molecule and acceptor molecule is r = 2.41 (r < 7) nm, which indicated that the non-radiative energy transfer from PCL to BSA [8,37].
-15
c -20
a -25 200
210
220
230
240
Wavelength (nm) Fig. 7. Circular dichroism (CD) spectrum of BSA with different molar ratios of PCL to BSA, (a–c): 0, 1:1, 5:1; [BSA]=0.20 lmol L1; T = 293 K.
a-helixð%Þ ¼
MRE208 4000 100 33; 000 4000
ð11Þ
where MRE208 is the observed MRE value at 208 nm, 4000 is the MRE of the b-form and random coil conformation cross at 208 nm and 33000 is the MRE value of a pure a-helix at 208 nm. From Eqs. (10) and (11), the a-helicity in the secondary structure of BSA can be determined. In this experiment, Cp = 0.20 lmol L1, n = 583, l = 1.0 cm. According to the above equations and experimental data, the values of a-helix (%) were 56.50% in free BSA, 54.90% in the PCL-BSA complex (molar ratio of PCL to BSA 1:1) and 50.50% with molar ratio 5:1, respectively. The data were indicative of the loss of a-helix due to the interaction of PCL with BSA. The percentage of protein ahelix structure decreased indicated that PCL bound with the amino acid residue of the main polypeptide chain of BSA and destroyed their hydrogen bonding networks, which indicated that PCL has changed the secondary structure of BSA [46]. Conclusions This paper investigated the interaction between BSA and PCL using fluorescence, UV–Vis and CD techniques under imitated physiological conditions. The results revealed that PCL could bind with BSA and the probable quenching mechanism of fluorescence of BSA by PCL was static. The binding constants, binding sites were obtained. What is more, the corresponding thermodynamic parameters at different temperatures were calculated according to Van’t Hoff equation, these data indicated that the hydrogen binds and
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