- Email: [email protected]
Interaction between a novel intramolecular charge transfer fluorescent probe PFEP and human serum albumin
Available online at www.sciencedirect.com
Chinese Chemical Letters 21 (2010) 480–483 www.elsevier.com/locate/cclet
Interaction between a novel intramolecular charge transfer fluorescent probe PFEP and human serum albumin Dong Ju, Sheng Mei Song, Yan Bo Wu, Shao Min Shuang, Chuan Dong * Research Center of Environmental Science and Engineering, College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China Received 17 August 2009
Abstract The interaction mechanism between human serum albumin (HSA) and 1-phenyl-3(fluorenone-2-yl)-5-(9-ethylcarbazole-3-yl)-2pyrazoline (PFEP) was investigated by fluorescence and absorption titration techniques in combination with molecular modeling method. Stern–Volmer plots at different temperatures proved that PFEP could quench the intrinsic fluorescence of HSA attributed to a static quenching procedure. The association constants were calculated in the range of 1 105–8 105 mol 1 at different pH conditions, and the stoichiometric ratio of binding was 1:1 between PEEP and HSA. Molecular modeling study showed that the ˚ in the optimal model. distance between indole moiety of the Trp214 residue and the carbazole group at the terminal of PFEP was 4.45 A # 2009 Chuan Dong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Fluorescent probe; Human serum albumin; ICT
1. Introduction As the most widely studied protein abundant in human blood plasma, researchers have made their efforts to try various ways to detect human serum albumin (HSA) for qualitative and quantitative analysis. It is significant not only in understanding the mechanism of interaction, but also for guiding the design of new drugs [1]. Molecules undergo an intramolecular charge transfer (ICT) reaction in the first excited singlet state with the resulting ICT emission being sensitive to changes in the external environment which produces the dramatic fluorescence spectral changes [2,3]. To further extend ICT molecules, a high sensitivity ICT fluorescent probe: 1-phenyl-3(fluorenone-2-yl)-5-(9ethylcarbazole-3-yl)-2-pyrazoline (PFEP) was designed and synthesized in our previous research, which produced ultra fast process of ICT from the big estimated dipole moment (41.19 D) of PFEP [4] Fig. 1 showed the ICT process of PFEP. In this work, the structural information of PFEP and HSA under different pH environments was characterized by using absorption, FTIR and fluorescence spectroscopic approaches, respectively. The interaction mechanism between PFEP and HSA was systematically investigated, and the binding curve of PFEP to HSA was constructed. The binding mode of PFEP to HSA was proposed and discussed on the molecular modeling method.
* Corresponding author. E-mail address: [email protected] (C. Dong). 1001-8417/$ – see front matter # 2009 Chuan Dong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2009.11.042
D. Ju et al. / Chinese Chemical Letters 21 (2010) 480–483
481
Fig. 1. The molecular structure and ICT process of compound PFEP.
2. Experimental PEEP was synthesized and purified as described in the literature [4]. Its stock standard solution with the concentration of 1.0 10 2 mol/L was prepared by directly dissolving in DMSO. HSA was commercially obtained from Sigma Chemical Co. (USA). It was dissolved in NaCl (0.1 mol/L) solution at a final concentration of 2.5 10 4 mol/L and stored at 0–4 8C. A series of HSA solutions (2.0 10 6 mol/L) were prepared by diluting the stock solution with Britton–Robinson buffer solution at different pH values. The absorption spectra were obtained by using TU-1901 spectrophotometer (Beijing Purkinje General Instrument Co. Ltd., China) equipped with 1.0 cm quartz cells. Intrinsic fluorescence quenching spectrum between 300 and 500 nm were recorded on Cary Eclipse spectrofluorometer (Varian, Japan) equipped with a 150W xenon lamp source and 1.0 cm quartz cell at excitation wavelengths of 295 and 280 nm. The measurement of pH value of solution was performed on a Model pHS-3C pH meter (Shanghai REX Instrument Factory, Shanghai, China). Molecular modeling study was performed on a SGI workstation using Insight II 2000 software where the pH value was set to 7.4. The potential of the 3D structures of HSA was assigned to the consistent valence force field (CVFF) which was often used to calculate polypeptide and protein. The PFEP compound was constructed in the Builder Module, while the crystal structure of the HSA was downloaded from the National Center for Biotechnology Information (NCBI) [5].
Fig. 2. UV–vis absorption spectra of HSA in the present of PFEP at pH 4.1, 7.4, 9.15 and 11.2. CPFEP(10 CHSA = 2.0 10 6 mol/L.
6
mol/L) = 0, 4, 8, 12, 16, 20 from 1 to 6,
482
D. Ju et al. / Chinese Chemical Letters 21 (2010) 480–483
Fig. 3. Fluorescence quenching spectra of HSA in the presence of PFEP at pH 4.1, 7.4, 9.15 and 11.2. lex = 295 nm. CPFEP(10 16, 20 from 1 to 6. Insert gives the linear fitting of (F0 F)/F against the concentration of the PFEP.
6
mol/L) = 0, 4, 8, 12,
3. Results and discussion Fig. 2 showed the UVabsorption spectra of HSA after interacting with PFEP under different pH conditions. It can be noted that the UV absorption band of HSA had an obvious hypsochromic shift, and the shifts gradually decreased when the pH of the buffer solutions changed from 11.20 to 4.10 indicating the specific interactions between PFEP and HSA. It can be seen from Fig. 3 that the fluorescence emission spectra had been dramatically quenched accompanying obviously red shift, signified that the surrounding environment of HSA had been changed as a result of its binding to PFEP. In the linear range of Stern–Volmer regression curves the average quenching constants for PEEP under various pH conditions were calculated and listed in Table 1. It could be seen that the bimolecular quenching constants (kq) were in the range of 1.2–1.8 1013 mol 1 s 1. These values are 103-fold higher than the maximum value possible for diffusion limited quenching in water ((about 2 1010 mol 1) [6], which suggested that the quenching was not initiated by dynamic collision but may arose from static quenching by complex formation [7]. No fluorescence emission was observed from PFEP in the range of 300–500 nm in aqueous solution, and the solvent DMSO had no effect on the fluorescence intensity of HSA, thus the contribution of PFEP and DMSO could be neglected. It could be calculated that J = 4.66 1013 cm 1 mol 1 nm4, R0 = 2.2 nm, E = 0.24 and r = 2.67 according to the Fo¨rster’s theory. The average distances were on the 2–8 nm scale and 0.5R0 < r < 1.5R0 [8], this indicated that the Table 1 Parameters of the PFEP–HSA complex at different pHa. pH
KSV(105 mol 1)
R
kq(1013 mol
3.29 4.10 5.02 6.09 7.4 8.36 9.15 10.38 11.2
1.717 0.057 1.37 0.02 1.456 0.062 1.201 0.021 1.206 0.032 1.343 0.052 1.360 0.035 1.568 0.042 1.716 0.037
0.99967 0.9998 0.99907 0.99912 0.99995 0.99982 0.99987 0.99997 0.99998
1.717 0.057 1.37 0.02 1.456 0.062 1.201 0.021 1.206 0.032 1.343 0.052 1.360 0.035 1.568 0.042 1.716 0.037
a
Average results of 5 experiments, lex = 295 nm, T = 25 8C.
1
s 1)
Ka(105 mol 1)
n
3.36 1.63 6.37 2.61 4.31 7.79 3.89 3.86 2.31
1.14 1.07 1.18 1.09 1.14 1.19 1.14 1.15 1.11
D. Ju et al. / Chinese Chemical Letters 21 (2010) 480–483
483
˚ of the ligand are displayed. Fig. 4. Interaction model between PFEP and HSA, only residues around 5 A
energy transfer from HSA to PFEP occurred with high probability, and larger PFEP–HSA distance r compared to that of R0 values in the present study also revealed the static quenching process [9]. Molecular modeling study was performed to identify the primary binding site that PFEP located. The results revealed that the optimal bonding model was the insertion of one arm of PFEP molecule into hydrophobic cavity of active site in subdomain IIA of HAS. As shown in Fig. 3, the N-ethylcarbazole group at the end of molecule PFEP had stacked with the Trp-214 residue inside the cavity [10]. The distance between indole moiety of the Trp214 residue and ˚ in the optimal model, which can facilitate the intermolecular the carbazole group at the terminal of PFEP was 4.45 A energy transfer to the PFEP group [11]. The hydrogen atoms except one of them involving hydrogen bonds are removed from HSA and PFEP. The residues of HSA are represented using black line (only Trp-214 is displayed in CPK style) and the structure of PFEP is represented using ball-and-stick model as seen in Fig. 4. The binding site of PFEP on HSA was determined by comparing the spectroscopic properties of PFEP after interacting with the protein in simulative physiological condition (pH 7.4) and those in buffer solution of pH 4.1. Their similar spectroscopic properties illustrated that PFEP was located in subdomain IIA in the vicinity of Trp-214 because the subdomain IIIA was unfolded in the F form of HSA (pH 4.1). It was suggested that the probe molecule was stabilized by the polar and ionic side chains of amino acid residues via hydrophobic association, van der Waals interactions, electrostatic interactions and H-bonds. Acknowledgment This work was supported by the National Natural Science Foundation of China (No. 20875059). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
T. Peters Jr., All About Albumin: Biochemistry, Genetics and Medical Application, Academic Press, San Diego, 1996. O. Sire, B. Alpert, C.A. Royer, Biophys. J. 70 (1996) 2903. M. Mazumdar, P.K. Parrack, B. Bhattacharyya, Eur. J. Biochem. 204 (1992) 127. S.M. Song, D. Ju, J.F. Li, Talanta 77 (2009) 1707. B.S. Sugio, A. Kashima, S. Mochizuki, et al. Protein Eng. 12 (1999) 439 http://www.ncbi.nlm.nih.gov/. W.R. Ware, J. Phys. Chem. 66 (1962) 455. J.R. Lakowicz, Principles of Fluorescence Spectroscopy, third ed., Plenum Press, New York, 2006. B. Valeur, Molecular Fluorescence: Principles and Applications, Wiley, New York, 2001. B. Valeur, J.C. Brochon, New Trends in Fluorescence Spectroscopy, 6th ed., Springer Press, Berlin, 1999. C. Qin, M.X. Xie, Y. Liu, Biomacromolecules 8 (2007) 2182. A. Sytnic, Y. Lritvinyuk, Proc. Natl. Acad. Sci. USA 93 (1996) 12959.
Chinese Chemical Letters 21 (2010) 480–483 www.elsevier.com/locate/cclet
Interaction between a novel intramolecular charge transfer fluorescent probe PFEP and human serum albumin Dong Ju, Sheng Mei Song, Yan Bo Wu, Shao Min Shuang, Chuan Dong * Research Center of Environmental Science and Engineering, College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China Received 17 August 2009
Abstract The interaction mechanism between human serum albumin (HSA) and 1-phenyl-3(fluorenone-2-yl)-5-(9-ethylcarbazole-3-yl)-2pyrazoline (PFEP) was investigated by fluorescence and absorption titration techniques in combination with molecular modeling method. Stern–Volmer plots at different temperatures proved that PFEP could quench the intrinsic fluorescence of HSA attributed to a static quenching procedure. The association constants were calculated in the range of 1 105–8 105 mol 1 at different pH conditions, and the stoichiometric ratio of binding was 1:1 between PEEP and HSA. Molecular modeling study showed that the ˚ in the optimal model. distance between indole moiety of the Trp214 residue and the carbazole group at the terminal of PFEP was 4.45 A # 2009 Chuan Dong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Fluorescent probe; Human serum albumin; ICT
1. Introduction As the most widely studied protein abundant in human blood plasma, researchers have made their efforts to try various ways to detect human serum albumin (HSA) for qualitative and quantitative analysis. It is significant not only in understanding the mechanism of interaction, but also for guiding the design of new drugs [1]. Molecules undergo an intramolecular charge transfer (ICT) reaction in the first excited singlet state with the resulting ICT emission being sensitive to changes in the external environment which produces the dramatic fluorescence spectral changes [2,3]. To further extend ICT molecules, a high sensitivity ICT fluorescent probe: 1-phenyl-3(fluorenone-2-yl)-5-(9ethylcarbazole-3-yl)-2-pyrazoline (PFEP) was designed and synthesized in our previous research, which produced ultra fast process of ICT from the big estimated dipole moment (41.19 D) of PFEP [4] Fig. 1 showed the ICT process of PFEP. In this work, the structural information of PFEP and HSA under different pH environments was characterized by using absorption, FTIR and fluorescence spectroscopic approaches, respectively. The interaction mechanism between PFEP and HSA was systematically investigated, and the binding curve of PFEP to HSA was constructed. The binding mode of PFEP to HSA was proposed and discussed on the molecular modeling method.
* Corresponding author. E-mail address: [email protected] (C. Dong). 1001-8417/$ – see front matter # 2009 Chuan Dong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2009.11.042
D. Ju et al. / Chinese Chemical Letters 21 (2010) 480–483
481
Fig. 1. The molecular structure and ICT process of compound PFEP.
2. Experimental PEEP was synthesized and purified as described in the literature [4]. Its stock standard solution with the concentration of 1.0 10 2 mol/L was prepared by directly dissolving in DMSO. HSA was commercially obtained from Sigma Chemical Co. (USA). It was dissolved in NaCl (0.1 mol/L) solution at a final concentration of 2.5 10 4 mol/L and stored at 0–4 8C. A series of HSA solutions (2.0 10 6 mol/L) were prepared by diluting the stock solution with Britton–Robinson buffer solution at different pH values. The absorption spectra were obtained by using TU-1901 spectrophotometer (Beijing Purkinje General Instrument Co. Ltd., China) equipped with 1.0 cm quartz cells. Intrinsic fluorescence quenching spectrum between 300 and 500 nm were recorded on Cary Eclipse spectrofluorometer (Varian, Japan) equipped with a 150W xenon lamp source and 1.0 cm quartz cell at excitation wavelengths of 295 and 280 nm. The measurement of pH value of solution was performed on a Model pHS-3C pH meter (Shanghai REX Instrument Factory, Shanghai, China). Molecular modeling study was performed on a SGI workstation using Insight II 2000 software where the pH value was set to 7.4. The potential of the 3D structures of HSA was assigned to the consistent valence force field (CVFF) which was often used to calculate polypeptide and protein. The PFEP compound was constructed in the Builder Module, while the crystal structure of the HSA was downloaded from the National Center for Biotechnology Information (NCBI) [5].
Fig. 2. UV–vis absorption spectra of HSA in the present of PFEP at pH 4.1, 7.4, 9.15 and 11.2. CPFEP(10 CHSA = 2.0 10 6 mol/L.
6
mol/L) = 0, 4, 8, 12, 16, 20 from 1 to 6,
482
D. Ju et al. / Chinese Chemical Letters 21 (2010) 480–483
Fig. 3. Fluorescence quenching spectra of HSA in the presence of PFEP at pH 4.1, 7.4, 9.15 and 11.2. lex = 295 nm. CPFEP(10 16, 20 from 1 to 6. Insert gives the linear fitting of (F0 F)/F against the concentration of the PFEP.
6
mol/L) = 0, 4, 8, 12,
3. Results and discussion Fig. 2 showed the UVabsorption spectra of HSA after interacting with PFEP under different pH conditions. It can be noted that the UV absorption band of HSA had an obvious hypsochromic shift, and the shifts gradually decreased when the pH of the buffer solutions changed from 11.20 to 4.10 indicating the specific interactions between PFEP and HSA. It can be seen from Fig. 3 that the fluorescence emission spectra had been dramatically quenched accompanying obviously red shift, signified that the surrounding environment of HSA had been changed as a result of its binding to PFEP. In the linear range of Stern–Volmer regression curves the average quenching constants for PEEP under various pH conditions were calculated and listed in Table 1. It could be seen that the bimolecular quenching constants (kq) were in the range of 1.2–1.8 1013 mol 1 s 1. These values are 103-fold higher than the maximum value possible for diffusion limited quenching in water ((about 2 1010 mol 1) [6], which suggested that the quenching was not initiated by dynamic collision but may arose from static quenching by complex formation [7]. No fluorescence emission was observed from PFEP in the range of 300–500 nm in aqueous solution, and the solvent DMSO had no effect on the fluorescence intensity of HSA, thus the contribution of PFEP and DMSO could be neglected. It could be calculated that J = 4.66 1013 cm 1 mol 1 nm4, R0 = 2.2 nm, E = 0.24 and r = 2.67 according to the Fo¨rster’s theory. The average distances were on the 2–8 nm scale and 0.5R0 < r < 1.5R0 [8], this indicated that the Table 1 Parameters of the PFEP–HSA complex at different pHa. pH
KSV(105 mol 1)
R
kq(1013 mol
3.29 4.10 5.02 6.09 7.4 8.36 9.15 10.38 11.2
1.717 0.057 1.37 0.02 1.456 0.062 1.201 0.021 1.206 0.032 1.343 0.052 1.360 0.035 1.568 0.042 1.716 0.037
0.99967 0.9998 0.99907 0.99912 0.99995 0.99982 0.99987 0.99997 0.99998
1.717 0.057 1.37 0.02 1.456 0.062 1.201 0.021 1.206 0.032 1.343 0.052 1.360 0.035 1.568 0.042 1.716 0.037
a
Average results of 5 experiments, lex = 295 nm, T = 25 8C.
1
s 1)
Ka(105 mol 1)
n
3.36 1.63 6.37 2.61 4.31 7.79 3.89 3.86 2.31
1.14 1.07 1.18 1.09 1.14 1.19 1.14 1.15 1.11
D. Ju et al. / Chinese Chemical Letters 21 (2010) 480–483
483
˚ of the ligand are displayed. Fig. 4. Interaction model between PFEP and HSA, only residues around 5 A
energy transfer from HSA to PFEP occurred with high probability, and larger PFEP–HSA distance r compared to that of R0 values in the present study also revealed the static quenching process [9]. Molecular modeling study was performed to identify the primary binding site that PFEP located. The results revealed that the optimal bonding model was the insertion of one arm of PFEP molecule into hydrophobic cavity of active site in subdomain IIA of HAS. As shown in Fig. 3, the N-ethylcarbazole group at the end of molecule PFEP had stacked with the Trp-214 residue inside the cavity [10]. The distance between indole moiety of the Trp214 residue and ˚ in the optimal model, which can facilitate the intermolecular the carbazole group at the terminal of PFEP was 4.45 A energy transfer to the PFEP group [11]. The hydrogen atoms except one of them involving hydrogen bonds are removed from HSA and PFEP. The residues of HSA are represented using black line (only Trp-214 is displayed in CPK style) and the structure of PFEP is represented using ball-and-stick model as seen in Fig. 4. The binding site of PFEP on HSA was determined by comparing the spectroscopic properties of PFEP after interacting with the protein in simulative physiological condition (pH 7.4) and those in buffer solution of pH 4.1. Their similar spectroscopic properties illustrated that PFEP was located in subdomain IIA in the vicinity of Trp-214 because the subdomain IIIA was unfolded in the F form of HSA (pH 4.1). It was suggested that the probe molecule was stabilized by the polar and ionic side chains of amino acid residues via hydrophobic association, van der Waals interactions, electrostatic interactions and H-bonds. Acknowledgment This work was supported by the National Natural Science Foundation of China (No. 20875059). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
T. Peters Jr., All About Albumin: Biochemistry, Genetics and Medical Application, Academic Press, San Diego, 1996. O. Sire, B. Alpert, C.A. Royer, Biophys. J. 70 (1996) 2903. M. Mazumdar, P.K. Parrack, B. Bhattacharyya, Eur. J. Biochem. 204 (1992) 127. S.M. Song, D. Ju, J.F. Li, Talanta 77 (2009) 1707. B.S. Sugio, A. Kashima, S. Mochizuki, et al. Protein Eng. 12 (1999) 439 http://www.ncbi.nlm.nih.gov/. W.R. Ware, J. Phys. Chem. 66 (1962) 455. J.R. Lakowicz, Principles of Fluorescence Spectroscopy, third ed., Plenum Press, New York, 2006. B. Valeur, Molecular Fluorescence: Principles and Applications, Wiley, New York, 2001. B. Valeur, J.C. Brochon, New Trends in Fluorescence Spectroscopy, 6th ed., Springer Press, Berlin, 1999. C. Qin, M.X. Xie, Y. Liu, Biomacromolecules 8 (2007) 2182. A. Sytnic, Y. Lritvinyuk, Proc. Natl. Acad. Sci. USA 93 (1996) 12959.