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Molecular simulation of the interaction between novel type rhodanine derivative probe and bovine serum albumin
Spectrochimica Acta Part A 74 (2009) 277–281
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Molecular simulation of the interaction between novel type rhodanine derivative probe and bovine serum albumin Jinghua Yu a,∗ , Bo Li b , Ping Dai a , Shenguang Ge a a b
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, Shandong Province, People’s Republic of China Tianjin Cement Industry Design and Research Institute Co., Ltd, Tianjin 300400, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 19 December 2008 Received in revised form 4 June 2009 Accepted 7 June 2009 Keywords: Fluorescence spectrum Rhodanine derivative Bovine serum albumin Interaction
a b s t r a c t The interaction between 3-(4 -methylphenyl)-5-(4 -methyl-2 -sulfophenylazo) rhodanine (M4MRASP) and bovine serum albumin (BSA) was studied by using spectrofluorimetry. It was shown in fluorescence spectrums that the quenching mechanism of BSA by M4MRASP was a static quenching. Meanwhile, the binding constant and binding site numbers were calculated. The action distance (r = 8.03 nm) and energy transfer efficiency (E = 0.12) between donor (BSA) and acceptor (M4MRASP) were obtained according to the theory of Förster non-radiation energy transfer. The effect of M4MRASP on the conformation of BSA was further analyzed by using synchronous fluorescence spectrometry. A new model of the interaction between small organic molecule and biomacromolecule was established. The results offered a reference for the studies on the biological effects and action mechanism of small molecule with protein. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Serum albumin, which can be combined with many endogenous and exogenous compounds, is most abundant in the plasma and plays an important role in storage and transport of energy [1]. It was very significant to investigate deeply the interaction between protein and small molecule to know the interaction mechanism of small molecule with protein at the molecular level. It is also important for research on proteomics, pharmacology, toxicology and the effect of environmental pollutants on living matter [2]. At present, considerable attention has been caused [3–6] to study bonding action of protein and small organic dye molecule. But mechanism of the interaction and equilibrium law of those bonding actions were still unclear and were being studied [7,8]. The phenylazo rhodanine reagents, which are big doublebond conjugated and have good planar structure, were often used in the determination of metal ions [9,10] with excellent performance as chromogenic and fluorescent reagents, higher sensitivities, good selectivities and stability, and so on, but hardly in the study of biomacromolecule. To improve the analytical performance, many rhodanine derivatives [11,12] with some active groups, such as sulfonic group, chloro group, nitryl and methoxyl groups, were synthesized. Yu [13] studied the spectral property of 3-(4 -methylphenyl)-5-(2 -sulfophenylazo) rhodanine as a probe with DNA and widened the research field of phenylazo rhodanine reagents. In this work, 3-(4 -methylphenyl)-
∗ Corresponding author. E-mail address: [email protected] (J.H. Yu.). 1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2009.06.013
5-(4 -methyl-2 -sulfophenylazo) rhodanine (M4MRASP, Fig. 1) was synthesized by the author and used as a spectral probe. At human physiological pH the interaction between bovine serum albumin (BSA) and M4MRASP was investigated detailedly by spectrofluorometry, synchronous spectrofluorometry and quantum chemistry. A novel model of interaction of protein with M4MRASP was established by calculating their binding constants, binding site numbers at different temperatures and space position, and also by studying the change of protein conformation. Those studies containing detailed and accurate data were very successful, which were beneficial to investigate the interaction mechanism of rhodanine reagents with protein and enlarge their application area. 2. Materials and methods 2.1. Apparatus and reagents A LS-55 fluorimeter (P-E, America) was used for recording the fluorescent spectra and measuring the intensity of fluorescence. A UV-3101PC spectrophotometer (Shimadzu, Japan) was used for recording the absorption spectra. A PHS-3C meter (Shang Hai Lei Ci Device Works, Shanghai, China) was used for pH measurement. The standard solution of M4MRASP (2.0 × 10−4 mol/L) was prepared by dissolving 0.0084 g M4MRASP in 100 mL doubledeionized water. The standard solution of BSA (1.0 × 10−5 mol/L) was obtained by dissolving 0.0335 g BSA in 50 mL double-deionized water. C-L buffer solution (pH 7.0) was prepared by mixing 0.2 mol/L KHC8 H4 O4 and 0.2 mol/L NaOH in suitable proportion, and the pH value was adjusted to 7 on the pH meter. All reagents were of
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J. Yu et al. / Spectrochimica Acta Part A 74 (2009) 277–281
Fig. 1. The structure of M4MRASP.
analytical reagent grade and above. Double-deionized water was used throughout. 2.2. Procedure Solutions were added in a calibrated flask in order of 1.0 mL 1.0 × 10−5 mol/L BSA, 2.5 mL C-L buffer solution (pH 7.0) and appropriate amounts of 2.0 × 10−4 mol/L M4MRASP solution, then diluted to 10 mL with water and mixed well. Then fluorescence quenching spectrums of BSA were obtained with exitation wavelength at 282 nm and emission wavelength at 300–490 nm. In addition, synchronous fluorescence spectrums of tryptophan residue and tyrosine residue were obtained at = 60 nm and 15 nm, respectively and UV absorption spectrums of M4MRSP were recorded at wavelength range of 300–500 nm. 3. Results and discussion 3.1. The quenching mechanism The intrinsic fluorescence of BSA comes from tryptophan and tyrosine residues. The interaction of protein with other molecules can result in the decline of its fluorescence intensity, and the phenomenon is called fluorescence quenching. There are two types of fluorescence quenching: dynamic and static quenching. Static quenching often generates from complex reactions in which nonfluorescence substance is obtained and this can affect secondary structure and physiological activity of protein while dynamic quenching does not do, in which transfer of energy or electron occurs. According to the procedure, fluorescence quenching spectrums of BSA at 293 K and 303 K were obtained (Figs. 2 and 3).
Fig. 3. Quenching of BSA fluorescence spectrums on adding M4MRASP (303 K) (1–9) M4MRASP (×10−5 mol/L) 0, 1.0, 1.2, 1.4, 1.8, 2.0, 2.4, 2 8. 3.0. BSA, 10−6 mol/L.
It could be found from Figs. 2 and 3, the fluorescence maximum wavelength of BSA and M4MRASP were 333 nm and 380 nm, respectively. When BSA and M4MRASP were mixed, a new fluorescence peak occurred at 370 nm and was not very obvious, but the intensity of the peak increased gradually and showed a red shift with the addition of M4MRASP, meanwhile, an equal emission point at 351 nm was also found with the addition of M4MRASP. The fluorescence intensities were quenched and enhanced before and after equal emission point respectively, but some curves of fluorescence peaks were overwritten by those of the adjacent fluorescence peaks, which indicated the additional quantity of M4MRASP was not the only factor of quenching. As a whole, these spectral phenomena showed a reaction that occurred between M4MRASP and BSA to form M4MRASP–BSA complex. It was assumed dynamic quenching occurred, and the mechanism of M4MRASP with BSA was investigated according to Stern-Volmer [14] equation which was used for the analysis of dynamic quenching. F0 = 1 + Kq 0 [Rt ] = 1 + KSV [Rt ] F
(1)
where F0 and F are the steady-state fluorescence intensities in the absence and presence of quencher, respectively, Kq is the quenching rate constant of the biomolecule, 0 is the average fluorescence lifetime of biomolecule without quencher, KSV is the Stern-Volmer quenching constant and [Rt ] is the concentration of quencher. The average fluorescence lifetime of biomolecule is 10−8 s [14]. The standard curve was made with the [Rt ] as abscissa and F0 /F as vertical coordinate (see Figs. 4 and 5) and Stern-Volme linear equation, linear correlation coefficient r, KSV and Kq were calculated and listed in Table 1. It could be found from Table 1 that KSV values decreased with temperature went higher, which was consistent with the static type of quenching mechanism and Kq values at 293 K and 303 K were 1.60 × 1012 and 1.53 × 1012 L/(mol s), respectively, which were both greater obviously than the Kq of the scatter procedure. This proved that the quenching type was static. 3.2. The binding constants and binding sites of M4MRASP with BSA The quantitative relationship between fluorescence quenching intensities and the concentration of quencher was obtained by the formula [15] as follows:
Fig. 2. Quenching of BSA fluorescence spectrums on adding M4MRASP (295 K). (1–9) M4MRASP (×10−5 mol/L) 0, 1.0, 1.2, 1.4, 1.8, 2.0. 2.4, 2.8, 3.0. BSA, 10−6 mol/L. (10) M4MRASP 2.0 × 10−5 mol/L.
lg
F − F 0 F
= lg K + n lg [Rt ]
(2)
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Table 1 Regression equations and interrelated constants at different temperatures. T (K)
Regression equations
295 303
F0 /F = 1.01 + 1.60 × 10 [Rt ] F0 /F = 1.01 + 1.53 × 104 [Rt ] 4
Linear correlation coefficient, r
KSV (L/mol)
Kq [L/(mol s)]
0.994 0.995
1.60 × 10 1.53 × 104
1.60 × 1012 1.53 × 1012
4
Table 2 Linear equations, correlation coefficients, binding constants and binding numbers. T (K)
Regression equations
Linear correlation coefficient, r
KSV (L/mol)
n
295 303
lg[(F0 − F)/F] = 3.83 + 0.92 lg [Rt ] lg[(F0 − F)/F] = 3.64 + 0.88 lg [Rt ]
0992 0.994
6.76 × 103 4.37 × 103
0.92 0.88
where K and n are binding constant and binding site numbers, respectively. Data from the fluorescence quenching spectrums (Figs. 2 and 3) was analyzed according to formula (2) and linear equations of binding constant and binding site numbers at 293 K and 303 K were obtained with the lg[Rt ] as abscissa and lg[(F0 − F)/F] as vertical coordinate. In addition, binding constant K and binding site numbers n were listed in Table 2. A higher temperature results in faster diffusion and larger amounts of collisional quenching, and will typically lead to the dissociation of weakly bound complexes and smaller amounts of static quenching [16]. Therefore, the binding number increases and decreases with the temperature increased for dynamic and static quenching, respectively.
As could be seen from Table 2, binding constant and binding site numbers both declined with the increase of the temperature, which further proved that the mechanism of M4MRASP with BSA was static quenching. 3.3. Investigation of binding mode The acting forces by which a small molecule can bond to biomacromoleclue mainly include Van Der Waals’ force and hydrogen bond, electrostatic force, hydrophobic force. The acting force types were investigated by calculating the thermodynamic parameters, enthalpy (r Hm ), entropy (r Sm ) and free energy change (r Gm ) from the following equations. 2.303RT1 T2 K2 lg T2 − T1 K1
(3)
r Gm = −RT ln K = r Hm − Tr Sm
(4)
r Hm =
The thermodynamic parameters were listed in Table 3. Ross and Subramanian [17] summarized in detail the relationship between acting force and thermodynamic parameters, when H > 0, S > 0, the acting force was hydrophobic force, when H < 0, S < 0, it was Van Der Waals’ force and hydrogen bond, and when H < 0, S > 0, it was electrostatic force. As could be seen from Table 3, the fact r Gm < 0 proved that the reaction was spontaneous, and that H < 0, S > 0 proved the acting force type was Van Der Waals’ force and hydrogen bond. 3.4. Molecular interaction distance of M4MRASP with tryptophan residues in BSA Fig. 4. Stem-Volme linear equation (295 K).
According to the theory of Förster non-radiation energy transfer [18], the condition in which non-radiation energy transfer can occur, including (1) donor is a kind of fluorescent material, (2) the distance between donor and acceptor should be no more than 7 nm, (3) fluorescence spectrum of donor overlaps well with absorption spectrum of acceptor. It can be forecasted from overlap of the fluorescence spectrum of donor (BSA) and absorption spectrum of acceptor (M4MRASP) in Fig. 6 that energy transfer between BSA and M4MRASP is likely to exist. According to the theory of Förster non-radiation energy transfer, the distance between the fluorescence emission group in donor (BSA) and binding site was determined. Based on the following
Table 3 Thermodynamic parameters of the binding reaction.
Fig. 5. Stern-Volme linear equation (303 K).
T (K)
r Gm (J/mol)
r Hm (J/mol)
r Sm [J/(mol K)]
295 303
−21,629.5 −21,114.0
−40,647.3 −40,647.3
−64.4672 −64.4665
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J. Yu et al. / Spectrochimica Acta Part A 74 (2009) 277–281
Fig. 7. Binding diagram of tryptophan residue (I) and M4MRASP (II). Fig. 6. Fluorescence spec trams of BSA and absorption spectrum of M4MRASP (l) cBSA = 10−6 mol/L, (2) cM4MRASP = 10−5 mol/L.
relationships, all parameters were calculated. E =1−
R6 F = 6 0 F0 R0 + r 6
R06 = 8.8 × 10−25 K 2 n−4 ϕJ
J=
F()ε()4
F()
(5) (6) (7)
where E is the efficiency of energy transfer, R0 is the critical energy transfer distance when the transfer efficiency E is 50%, r is the distance between the donor and the acceptor, K2 is the spatial orientation factor of the dipole, n is the refractive index of the medium, ϕ is the fluorescence quantum yield of the donor, J is the overlapped integral of the fluorescence emission spectrum of donor and the absorption spectrum of acceptor. F() is the fluorescence intensity of the fluorescent donor and ε() is the molarabsorptivity at wavelength . In the study of protein quenching, often K2 = 2/3, ϕ = 0.15, n = 1.336 [19], therefore E, J, R0 , and r were calculated to be 0.12, 1.34 × 1013 L nm4 /(mol cm), 5.77 nm and 8.03 nm, respectively. 3.5. Possible structure of BSA–M4MRASP complex BSA contains three homologous domains, I, II and III, and each domain can be divided into two subdomains, A and B. The investigation of the three-dimensional structure of serum albumin showed that IIA and IIIA are the main binding sites of small molecule with serum albumin. The fluorescence of protein mainly comes from tryptophan residues due to very efficient energy transfer among amino acid residues. There are two tryptophan residues, Trp-134 (in the subdomain IA) and Trp-212 (in the subdomain IIA) in each BSA molecule and the fluorescence of BSA mainly comes from Trp212 [18]. Therefore, the interaction between Trp-212 and M4MRASP mainly resulted in the fluorescence quenching of BSA, and Trp-212 was main binding site with M4MRASP. Based on the results of thermodynamic parameters, the acting force type was Van Der Waals’ force and hydrogen bond. Further study was carried on by quantum chemistry to determine the binding atom. The ground state charges and dipole moments of M4MRASP and Trp-214 were calculated by analytic grads method (RHF/3-21G*) of quantum chemistry using Gaussian-98 programe. The result showed that the hydrogen atom of sulfonic group in the M4MRASP molecule had a large positive charge density (0.471) and a small steric hindrance, it easily formed a hydrogen bond with the oxygen atom of carbonyl group in the tryptophan residue, which had a large negative charge density (−0.737). The hydrogen atom of amino group in the tryptophan residue had a small charge density (0.243) and a large steric hindrance, not easily forming a hydrogen bond with the oxygen atoms
of sulfonic group in the M4MRASP molecule. In addition, the dipole moment values of M4MRASP and tryptophan were 5.282 debye and 7.338 debye, and they were very large, which showed that the Van Der Waals’ force was strong. The analysis results with quantum chemistry and thermodynamic parameters were the same. Therefore, the binding type of tryptophan residue and M4MRASP was possible as follows (see Fig. 7). 3.6. The effect of M4MRASP on the conformation of BSA The maximum emission wavelengths of tryptophan residue and tyrosine residue in the protein molecule are related to the polarity of their surrounding, changes of the maximum emission wavelengths can reflect changes of protein conformation. Synchronous fluorescence spectrum, which reflects the change of the minimal structure of protein molecule with a narrow spectral band, high sensitivity and good selectivity, was often used in the study of protein conformation. Synchronous fluorescence spectrums of tryptophan residue and tyrosine residue were obtained at = 60 nm and 15 nm, respectively [20] (Figs. 8 and 9). As could be seen from Figs. 8 and 9, the fluorescence intensities of tryptophan residue and tyrosine residue decreased with the increase of the concentration of M4MRASP. Tryptophan residue, the fluorescence intensities of which were stronger than those of tyrosine residue, was the main source of fluorescence. The maximum emission wavelength of tyrosine residue had a blue shift and tryptophan residue had a little red shift. Those spectral phenomenons
Fig. 8. Effect of M4MRASP on the synchronous fluorescence spectra of BAS ( = 15 nm).
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the study of the interaction of small molecule with protein. Therefore, the investigation between phenylazo rhodanine derivatives and protein will be emphasized to establish the structure–activity relationship between probes with different structures and the binding performance with protein, and provide much powerful support for selection and design of drug molecules. Acknowledgements This work is financially supported by Natural Science Research Foundation of Shandong Province, China (Y2007B07), Postgraduate Innovation Program of Shandong Province (SDYY08030) and Key Subject Foundation of Shandong Province, China (XTD0705). References
Fig. 9. Effect of M4MRASP on the synchronous fluorescence spectra of BAS ( = 60 nm).
showed that addition of M4MRASP resulted in the change of protein conformation. 4. Prospect At present, the study of the interaction of sole small molecule with protein is very active, but it is lack of connection and comparison with each other. A series of phenylazo rhodanine derivatives were synthesized by the author, which had different substituent group and substitution position and the fluorescence properties of those derivatives had been investigated by both theory and experiments. In this paper, it is proved that M4MRASP is also a fine probe in
[1] R.H. Mcmenamy, Albumin Structure, Function and Uses, Degamon Press, Oxford, 1977. [2] B. Mauricios, L.I. Guilherme, J. Phys. Chem. B. 102 (1998) 4678–4688. [3] Q.Y. Zhou, Y. Yu, Chin. J. Anal. Chem. 31 (2003) 976–980. [4] J.Y. Fu, I. Mori, M. Toyoda, Chem. Pharm. Bull. 38 (1990) 956–959. [5] W. Yao, F. Gao, L. Wang, Chin. J. Sp. Lab. 23 (2006) 270–273. [6] G.X. Li, J.Q. Li, Y.X. Wei, C. Dong, Spectrosc. Spectral Anal. 25 (2005) 1277–1280. [7] P.G. Yi, J.F. Liu, Z.C. Shang, Q.S. Yu, Spectrosc. Spectral Anal. 21 (2001) 826–828. [8] C.C. Guo, H.P. Li, X.B. Zhang, R.B. Tong, Chem. J. Chin. Univ. 24 (2003) 282–287. [9] Y.C. Cui, Y.X. Yu, C.L. Yan, J.Z. Zhu, Chin. J. Sp. Lab. 24 (2007) 646–648. [10] J.H. Yu, Q.Y. Ou, Y. Lu, L.G. Wang, T. Li, Spectrosc. Spectral Anal. 24 (2004) 1093–1095. [11] J.H. Yu, S.G. Ge, Y. Tan, B. Li, X.L. Cheng, P. Dai, Chin. J. Anal. Chem. 36 (2008) 549–552. [12] J.H. Yu, B. Li, S.G. Ge, Y.L. Ding, Chin. J. Anal. Chem. 36 (2008) 866. [13] J. Yu, B. Li, Y. Zhu, X. Cheng, L. Zhang, J. Lumin. 23 (2008) 106. [14] E.L. Gelamo, C. Silva, H. Imasato, M. Tabak, BBA 1594 (2002) 84–99. [15] Y.P. Wang, Y.L. Wei, C. Dong, J. Photochem. Photobiol. A 177 (2006) 6–11. [16] F.L. Cui, J.L. Wang, Y.R. Cui, X.J. Yao, G.R. Qu, Y. Lu, J. Lumin. 22 (2007) 546–553. [17] P.D. Ross, S. Subramanian, Biochemistry 20 (1981) 3096–3102. [18] T. Förster, Modern Quantum Chemistry, vol. 3, Academic Press, New York, 1996. [19] M.M. Yang, P. Yang, L.W. Zhang, Chin. Sci. Bull. 39 (1994) 31–35. [20] C.Q. Ma, K.A. Li, F.L. Zhao, S.Y. Tong, Acta Chim. Sin. 57 (1999) 389–395.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Molecular simulation of the interaction between novel type rhodanine derivative probe and bovine serum albumin Jinghua Yu a,∗ , Bo Li b , Ping Dai a , Shenguang Ge a a b
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, Shandong Province, People’s Republic of China Tianjin Cement Industry Design and Research Institute Co., Ltd, Tianjin 300400, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 19 December 2008 Received in revised form 4 June 2009 Accepted 7 June 2009 Keywords: Fluorescence spectrum Rhodanine derivative Bovine serum albumin Interaction
a b s t r a c t The interaction between 3-(4 -methylphenyl)-5-(4 -methyl-2 -sulfophenylazo) rhodanine (M4MRASP) and bovine serum albumin (BSA) was studied by using spectrofluorimetry. It was shown in fluorescence spectrums that the quenching mechanism of BSA by M4MRASP was a static quenching. Meanwhile, the binding constant and binding site numbers were calculated. The action distance (r = 8.03 nm) and energy transfer efficiency (E = 0.12) between donor (BSA) and acceptor (M4MRASP) were obtained according to the theory of Förster non-radiation energy transfer. The effect of M4MRASP on the conformation of BSA was further analyzed by using synchronous fluorescence spectrometry. A new model of the interaction between small organic molecule and biomacromolecule was established. The results offered a reference for the studies on the biological effects and action mechanism of small molecule with protein. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Serum albumin, which can be combined with many endogenous and exogenous compounds, is most abundant in the plasma and plays an important role in storage and transport of energy [1]. It was very significant to investigate deeply the interaction between protein and small molecule to know the interaction mechanism of small molecule with protein at the molecular level. It is also important for research on proteomics, pharmacology, toxicology and the effect of environmental pollutants on living matter [2]. At present, considerable attention has been caused [3–6] to study bonding action of protein and small organic dye molecule. But mechanism of the interaction and equilibrium law of those bonding actions were still unclear and were being studied [7,8]. The phenylazo rhodanine reagents, which are big doublebond conjugated and have good planar structure, were often used in the determination of metal ions [9,10] with excellent performance as chromogenic and fluorescent reagents, higher sensitivities, good selectivities and stability, and so on, but hardly in the study of biomacromolecule. To improve the analytical performance, many rhodanine derivatives [11,12] with some active groups, such as sulfonic group, chloro group, nitryl and methoxyl groups, were synthesized. Yu [13] studied the spectral property of 3-(4 -methylphenyl)-5-(2 -sulfophenylazo) rhodanine as a probe with DNA and widened the research field of phenylazo rhodanine reagents. In this work, 3-(4 -methylphenyl)-
∗ Corresponding author. E-mail address: [email protected] (J.H. Yu.). 1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2009.06.013
5-(4 -methyl-2 -sulfophenylazo) rhodanine (M4MRASP, Fig. 1) was synthesized by the author and used as a spectral probe. At human physiological pH the interaction between bovine serum albumin (BSA) and M4MRASP was investigated detailedly by spectrofluorometry, synchronous spectrofluorometry and quantum chemistry. A novel model of interaction of protein with M4MRASP was established by calculating their binding constants, binding site numbers at different temperatures and space position, and also by studying the change of protein conformation. Those studies containing detailed and accurate data were very successful, which were beneficial to investigate the interaction mechanism of rhodanine reagents with protein and enlarge their application area. 2. Materials and methods 2.1. Apparatus and reagents A LS-55 fluorimeter (P-E, America) was used for recording the fluorescent spectra and measuring the intensity of fluorescence. A UV-3101PC spectrophotometer (Shimadzu, Japan) was used for recording the absorption spectra. A PHS-3C meter (Shang Hai Lei Ci Device Works, Shanghai, China) was used for pH measurement. The standard solution of M4MRASP (2.0 × 10−4 mol/L) was prepared by dissolving 0.0084 g M4MRASP in 100 mL doubledeionized water. The standard solution of BSA (1.0 × 10−5 mol/L) was obtained by dissolving 0.0335 g BSA in 50 mL double-deionized water. C-L buffer solution (pH 7.0) was prepared by mixing 0.2 mol/L KHC8 H4 O4 and 0.2 mol/L NaOH in suitable proportion, and the pH value was adjusted to 7 on the pH meter. All reagents were of
278
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Fig. 1. The structure of M4MRASP.
analytical reagent grade and above. Double-deionized water was used throughout. 2.2. Procedure Solutions were added in a calibrated flask in order of 1.0 mL 1.0 × 10−5 mol/L BSA, 2.5 mL C-L buffer solution (pH 7.0) and appropriate amounts of 2.0 × 10−4 mol/L M4MRASP solution, then diluted to 10 mL with water and mixed well. Then fluorescence quenching spectrums of BSA were obtained with exitation wavelength at 282 nm and emission wavelength at 300–490 nm. In addition, synchronous fluorescence spectrums of tryptophan residue and tyrosine residue were obtained at = 60 nm and 15 nm, respectively and UV absorption spectrums of M4MRSP were recorded at wavelength range of 300–500 nm. 3. Results and discussion 3.1. The quenching mechanism The intrinsic fluorescence of BSA comes from tryptophan and tyrosine residues. The interaction of protein with other molecules can result in the decline of its fluorescence intensity, and the phenomenon is called fluorescence quenching. There are two types of fluorescence quenching: dynamic and static quenching. Static quenching often generates from complex reactions in which nonfluorescence substance is obtained and this can affect secondary structure and physiological activity of protein while dynamic quenching does not do, in which transfer of energy or electron occurs. According to the procedure, fluorescence quenching spectrums of BSA at 293 K and 303 K were obtained (Figs. 2 and 3).
Fig. 3. Quenching of BSA fluorescence spectrums on adding M4MRASP (303 K) (1–9) M4MRASP (×10−5 mol/L) 0, 1.0, 1.2, 1.4, 1.8, 2.0, 2.4, 2 8. 3.0. BSA, 10−6 mol/L.
It could be found from Figs. 2 and 3, the fluorescence maximum wavelength of BSA and M4MRASP were 333 nm and 380 nm, respectively. When BSA and M4MRASP were mixed, a new fluorescence peak occurred at 370 nm and was not very obvious, but the intensity of the peak increased gradually and showed a red shift with the addition of M4MRASP, meanwhile, an equal emission point at 351 nm was also found with the addition of M4MRASP. The fluorescence intensities were quenched and enhanced before and after equal emission point respectively, but some curves of fluorescence peaks were overwritten by those of the adjacent fluorescence peaks, which indicated the additional quantity of M4MRASP was not the only factor of quenching. As a whole, these spectral phenomena showed a reaction that occurred between M4MRASP and BSA to form M4MRASP–BSA complex. It was assumed dynamic quenching occurred, and the mechanism of M4MRASP with BSA was investigated according to Stern-Volmer [14] equation which was used for the analysis of dynamic quenching. F0 = 1 + Kq 0 [Rt ] = 1 + KSV [Rt ] F
(1)
where F0 and F are the steady-state fluorescence intensities in the absence and presence of quencher, respectively, Kq is the quenching rate constant of the biomolecule, 0 is the average fluorescence lifetime of biomolecule without quencher, KSV is the Stern-Volmer quenching constant and [Rt ] is the concentration of quencher. The average fluorescence lifetime of biomolecule is 10−8 s [14]. The standard curve was made with the [Rt ] as abscissa and F0 /F as vertical coordinate (see Figs. 4 and 5) and Stern-Volme linear equation, linear correlation coefficient r, KSV and Kq were calculated and listed in Table 1. It could be found from Table 1 that KSV values decreased with temperature went higher, which was consistent with the static type of quenching mechanism and Kq values at 293 K and 303 K were 1.60 × 1012 and 1.53 × 1012 L/(mol s), respectively, which were both greater obviously than the Kq of the scatter procedure. This proved that the quenching type was static. 3.2. The binding constants and binding sites of M4MRASP with BSA The quantitative relationship between fluorescence quenching intensities and the concentration of quencher was obtained by the formula [15] as follows:
Fig. 2. Quenching of BSA fluorescence spectrums on adding M4MRASP (295 K). (1–9) M4MRASP (×10−5 mol/L) 0, 1.0, 1.2, 1.4, 1.8, 2.0. 2.4, 2.8, 3.0. BSA, 10−6 mol/L. (10) M4MRASP 2.0 × 10−5 mol/L.
lg
F − F 0 F
= lg K + n lg [Rt ]
(2)
J. Yu et al. / Spectrochimica Acta Part A 74 (2009) 277–281
279
Table 1 Regression equations and interrelated constants at different temperatures. T (K)
Regression equations
295 303
F0 /F = 1.01 + 1.60 × 10 [Rt ] F0 /F = 1.01 + 1.53 × 104 [Rt ] 4
Linear correlation coefficient, r
KSV (L/mol)
Kq [L/(mol s)]
0.994 0.995
1.60 × 10 1.53 × 104
1.60 × 1012 1.53 × 1012
4
Table 2 Linear equations, correlation coefficients, binding constants and binding numbers. T (K)
Regression equations
Linear correlation coefficient, r
KSV (L/mol)
n
295 303
lg[(F0 − F)/F] = 3.83 + 0.92 lg [Rt ] lg[(F0 − F)/F] = 3.64 + 0.88 lg [Rt ]
0992 0.994
6.76 × 103 4.37 × 103
0.92 0.88
where K and n are binding constant and binding site numbers, respectively. Data from the fluorescence quenching spectrums (Figs. 2 and 3) was analyzed according to formula (2) and linear equations of binding constant and binding site numbers at 293 K and 303 K were obtained with the lg[Rt ] as abscissa and lg[(F0 − F)/F] as vertical coordinate. In addition, binding constant K and binding site numbers n were listed in Table 2. A higher temperature results in faster diffusion and larger amounts of collisional quenching, and will typically lead to the dissociation of weakly bound complexes and smaller amounts of static quenching [16]. Therefore, the binding number increases and decreases with the temperature increased for dynamic and static quenching, respectively.
As could be seen from Table 2, binding constant and binding site numbers both declined with the increase of the temperature, which further proved that the mechanism of M4MRASP with BSA was static quenching. 3.3. Investigation of binding mode The acting forces by which a small molecule can bond to biomacromoleclue mainly include Van Der Waals’ force and hydrogen bond, electrostatic force, hydrophobic force. The acting force types were investigated by calculating the thermodynamic parameters, enthalpy (r Hm ), entropy (r Sm ) and free energy change (r Gm ) from the following equations. 2.303RT1 T2 K2 lg T2 − T1 K1
(3)
r Gm = −RT ln K = r Hm − Tr Sm
(4)
r Hm =
The thermodynamic parameters were listed in Table 3. Ross and Subramanian [17] summarized in detail the relationship between acting force and thermodynamic parameters, when H > 0, S > 0, the acting force was hydrophobic force, when H < 0, S < 0, it was Van Der Waals’ force and hydrogen bond, and when H < 0, S > 0, it was electrostatic force. As could be seen from Table 3, the fact r Gm < 0 proved that the reaction was spontaneous, and that H < 0, S > 0 proved the acting force type was Van Der Waals’ force and hydrogen bond. 3.4. Molecular interaction distance of M4MRASP with tryptophan residues in BSA Fig. 4. Stem-Volme linear equation (295 K).
According to the theory of Förster non-radiation energy transfer [18], the condition in which non-radiation energy transfer can occur, including (1) donor is a kind of fluorescent material, (2) the distance between donor and acceptor should be no more than 7 nm, (3) fluorescence spectrum of donor overlaps well with absorption spectrum of acceptor. It can be forecasted from overlap of the fluorescence spectrum of donor (BSA) and absorption spectrum of acceptor (M4MRASP) in Fig. 6 that energy transfer between BSA and M4MRASP is likely to exist. According to the theory of Förster non-radiation energy transfer, the distance between the fluorescence emission group in donor (BSA) and binding site was determined. Based on the following
Table 3 Thermodynamic parameters of the binding reaction.
Fig. 5. Stern-Volme linear equation (303 K).
T (K)
r Gm (J/mol)
r Hm (J/mol)
r Sm [J/(mol K)]
295 303
−21,629.5 −21,114.0
−40,647.3 −40,647.3
−64.4672 −64.4665
280
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Fig. 7. Binding diagram of tryptophan residue (I) and M4MRASP (II). Fig. 6. Fluorescence spec trams of BSA and absorption spectrum of M4MRASP (l) cBSA = 10−6 mol/L, (2) cM4MRASP = 10−5 mol/L.
relationships, all parameters were calculated. E =1−
R6 F = 6 0 F0 R0 + r 6
R06 = 8.8 × 10−25 K 2 n−4 ϕJ
J=
F()ε()4
F()
(5) (6) (7)
where E is the efficiency of energy transfer, R0 is the critical energy transfer distance when the transfer efficiency E is 50%, r is the distance between the donor and the acceptor, K2 is the spatial orientation factor of the dipole, n is the refractive index of the medium, ϕ is the fluorescence quantum yield of the donor, J is the overlapped integral of the fluorescence emission spectrum of donor and the absorption spectrum of acceptor. F() is the fluorescence intensity of the fluorescent donor and ε() is the molarabsorptivity at wavelength . In the study of protein quenching, often K2 = 2/3, ϕ = 0.15, n = 1.336 [19], therefore E, J, R0 , and r were calculated to be 0.12, 1.34 × 1013 L nm4 /(mol cm), 5.77 nm and 8.03 nm, respectively. 3.5. Possible structure of BSA–M4MRASP complex BSA contains three homologous domains, I, II and III, and each domain can be divided into two subdomains, A and B. The investigation of the three-dimensional structure of serum albumin showed that IIA and IIIA are the main binding sites of small molecule with serum albumin. The fluorescence of protein mainly comes from tryptophan residues due to very efficient energy transfer among amino acid residues. There are two tryptophan residues, Trp-134 (in the subdomain IA) and Trp-212 (in the subdomain IIA) in each BSA molecule and the fluorescence of BSA mainly comes from Trp212 [18]. Therefore, the interaction between Trp-212 and M4MRASP mainly resulted in the fluorescence quenching of BSA, and Trp-212 was main binding site with M4MRASP. Based on the results of thermodynamic parameters, the acting force type was Van Der Waals’ force and hydrogen bond. Further study was carried on by quantum chemistry to determine the binding atom. The ground state charges and dipole moments of M4MRASP and Trp-214 were calculated by analytic grads method (RHF/3-21G*) of quantum chemistry using Gaussian-98 programe. The result showed that the hydrogen atom of sulfonic group in the M4MRASP molecule had a large positive charge density (0.471) and a small steric hindrance, it easily formed a hydrogen bond with the oxygen atom of carbonyl group in the tryptophan residue, which had a large negative charge density (−0.737). The hydrogen atom of amino group in the tryptophan residue had a small charge density (0.243) and a large steric hindrance, not easily forming a hydrogen bond with the oxygen atoms
of sulfonic group in the M4MRASP molecule. In addition, the dipole moment values of M4MRASP and tryptophan were 5.282 debye and 7.338 debye, and they were very large, which showed that the Van Der Waals’ force was strong. The analysis results with quantum chemistry and thermodynamic parameters were the same. Therefore, the binding type of tryptophan residue and M4MRASP was possible as follows (see Fig. 7). 3.6. The effect of M4MRASP on the conformation of BSA The maximum emission wavelengths of tryptophan residue and tyrosine residue in the protein molecule are related to the polarity of their surrounding, changes of the maximum emission wavelengths can reflect changes of protein conformation. Synchronous fluorescence spectrum, which reflects the change of the minimal structure of protein molecule with a narrow spectral band, high sensitivity and good selectivity, was often used in the study of protein conformation. Synchronous fluorescence spectrums of tryptophan residue and tyrosine residue were obtained at = 60 nm and 15 nm, respectively [20] (Figs. 8 and 9). As could be seen from Figs. 8 and 9, the fluorescence intensities of tryptophan residue and tyrosine residue decreased with the increase of the concentration of M4MRASP. Tryptophan residue, the fluorescence intensities of which were stronger than those of tyrosine residue, was the main source of fluorescence. The maximum emission wavelength of tyrosine residue had a blue shift and tryptophan residue had a little red shift. Those spectral phenomenons
Fig. 8. Effect of M4MRASP on the synchronous fluorescence spectra of BAS ( = 15 nm).
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the study of the interaction of small molecule with protein. Therefore, the investigation between phenylazo rhodanine derivatives and protein will be emphasized to establish the structure–activity relationship between probes with different structures and the binding performance with protein, and provide much powerful support for selection and design of drug molecules. Acknowledgements This work is financially supported by Natural Science Research Foundation of Shandong Province, China (Y2007B07), Postgraduate Innovation Program of Shandong Province (SDYY08030) and Key Subject Foundation of Shandong Province, China (XTD0705). References
Fig. 9. Effect of M4MRASP on the synchronous fluorescence spectra of BAS ( = 60 nm).
showed that addition of M4MRASP resulted in the change of protein conformation. 4. Prospect At present, the study of the interaction of sole small molecule with protein is very active, but it is lack of connection and comparison with each other. A series of phenylazo rhodanine derivatives were synthesized by the author, which had different substituent group and substitution position and the fluorescence properties of those derivatives had been investigated by both theory and experiments. In this paper, it is proved that M4MRASP is also a fine probe in
[1] R.H. Mcmenamy, Albumin Structure, Function and Uses, Degamon Press, Oxford, 1977. [2] B. Mauricios, L.I. Guilherme, J. Phys. Chem. B. 102 (1998) 4678–4688. [3] Q.Y. Zhou, Y. Yu, Chin. J. Anal. Chem. 31 (2003) 976–980. [4] J.Y. Fu, I. Mori, M. Toyoda, Chem. Pharm. Bull. 38 (1990) 956–959. [5] W. Yao, F. Gao, L. Wang, Chin. J. Sp. Lab. 23 (2006) 270–273. [6] G.X. Li, J.Q. Li, Y.X. Wei, C. Dong, Spectrosc. Spectral Anal. 25 (2005) 1277–1280. [7] P.G. Yi, J.F. Liu, Z.C. Shang, Q.S. Yu, Spectrosc. Spectral Anal. 21 (2001) 826–828. [8] C.C. Guo, H.P. Li, X.B. Zhang, R.B. Tong, Chem. J. Chin. Univ. 24 (2003) 282–287. [9] Y.C. Cui, Y.X. Yu, C.L. Yan, J.Z. Zhu, Chin. J. Sp. Lab. 24 (2007) 646–648. [10] J.H. Yu, Q.Y. Ou, Y. Lu, L.G. Wang, T. Li, Spectrosc. Spectral Anal. 24 (2004) 1093–1095. [11] J.H. Yu, S.G. Ge, Y. Tan, B. Li, X.L. Cheng, P. Dai, Chin. J. Anal. Chem. 36 (2008) 549–552. [12] J.H. Yu, B. Li, S.G. Ge, Y.L. Ding, Chin. J. Anal. Chem. 36 (2008) 866. [13] J. Yu, B. Li, Y. Zhu, X. Cheng, L. Zhang, J. Lumin. 23 (2008) 106. [14] E.L. Gelamo, C. Silva, H. Imasato, M. Tabak, BBA 1594 (2002) 84–99. [15] Y.P. Wang, Y.L. Wei, C. Dong, J. Photochem. Photobiol. A 177 (2006) 6–11. [16] F.L. Cui, J.L. Wang, Y.R. Cui, X.J. Yao, G.R. Qu, Y. Lu, J. Lumin. 22 (2007) 546–553. [17] P.D. Ross, S. Subramanian, Biochemistry 20 (1981) 3096–3102. [18] T. Förster, Modern Quantum Chemistry, vol. 3, Academic Press, New York, 1996. [19] M.M. Yang, P. Yang, L.W. Zhang, Chin. Sci. Bull. 39 (1994) 31–35. [20] C.Q. Ma, K.A. Li, F.L. Zhao, S.Y. Tong, Acta Chim. Sin. 57 (1999) 389–395.