Chinese Journal of Chemistry, 2007, 25, 917—920 Full Paper

* E-mail: chemliup@mail.whut.edu.cn; Tel.: 0086-027-63410197; Fax: 0086-027-87651773 Received September 18, 2006; revised December 7, 2006; accepted March 26, 2007. Project supported by the National Natural Science Foundation of China (No. 30570015) and Open Fund of State Key Laboratory of Agricultural

Microbiology (Huazhong Agricultural University).

© 2007 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Study on the Interaction between Lanthanide Cationic Porphyrin Complex and Bovine Serum Albumin

LIU, Peng*,a(刘鹏) LIU, Yib(刘义) LI, Xia(李曦) HUANG, Wei-Guoc(黄伟国) a Department of Applied Chemistry, School of Sciences, Wuhan University of Technology, Wuhan, Hubei 430070,

China b College of Chemistry and Molecular Science, Wuhan University, Wuhan, Hubei 430072, China

c Department of Chemistry, Hong Kong Baptist University, Hong Kong, China

The interaction between lanthanide cationic porphyrin and bovine serum albumin (BSA) was studied by fluo-rescence and UV-Vis spectrum. The static quenching of BSA was observed in the presence of YbTMPyP. Accord-ing to the thermodynamic parameters, this binding was regarded as “enthalpy-driven” reaction. Furthermore, YbTMPyP is so close to the residues of BSA that molecular resonance energy transfer occurs between them. Be-sides, the red drift and hypochromicity of absorption spectrum of YbTMPyP were accompanied with the binding reaction.

Keywords porphyrin, BSA, fluorescence, enthalpy-driven, hypochromicity

Introduction

Porphyrins have been under intensive study with in-terest in its potential therapeutic use.1-3 By design of porphyrins with special structure, a series of porphyrins were synthesized and developed for the application to medicine. For example, it has been suggested that free porphyrin bases could interact with bio-molecules by various binding modes, including intercalation, outside stacking, outside random binding, and groove binding.4 Furthermore, porphyrins have been widely investigated as anti-cancer drugs in photodynamic therapy, in part because both cationic and anionic derivatives can be easily obtained and administered. Lanthanide cationic porphyrin complexes are known for their unique optical properties such as line-like emission and long lumines-cence lifetime.5 This property is important for photody-namic therapy in the area of medicine. YbTMPyP {Yb- [5,10,15,20-tetrakis(p-methylpyridine)porphyrin]} is a kind of water-soluble cationic lanthanide cationic por-phyrin complex, whose structure is shown in Figure 1. In this study, we try to reveal the interaction between YbTMPyP and BSA.

Serum albumin has been implicated in transport, storage, and metabolism of many metal ions, and has many physiological functions as the major constituents of the circulatory system.6-8 According to the analysis of amino acid sequence, there are 583 amino acids and 17 disulfide bridges in BSA, which contains about 55% α-helix content. BSA molecule is markedly polar with a potential for 100 negative and 82 positive charges at neutral pH.9,10 Many bioactive small molecules bind

Figure 1 The structure of YbTMPyP.

reversibly BSA and other serum components, which then work as carriers. Furthermore, BSA often increases the apparent solubility of hydrophobic drugs in plasma and modulates their delivery to cells in vivo and in vitro. For development of therapy function of porphyrins, it is important to study the interactions of the porphyrins with serum albumin.

Experimental

Materials

BSA with a purity of no less than 99.5% was pur-chased from Sigma, and dissolved in Tris-HCl buffer solution. YbTMPyP was prepared according to litera-ture procedure.5,11 All solutions were prepared with

918 Chin. J. Chem., 2007, Vol. 25, No. 7 LIU et al.

© 2007 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

doubly distilled water.

Instrument

All fluorescence spectra were recorded on an F-2500 spectrofluorimeter in the ratio mode with temperature maintained by circulating bath (Hitachi, Japan). TU- 1901 spectrophotometer (Puxi Ltd. of Beijing, China) was used for scanning UV-Vis spectra. And the mass of the sample was accurately weighed using a microbal-ance (Sartorius, ME215S) with a resolution of 0.1 mg.

Spectroscopic measurements

The absorption spectra of BSA, YbTMPyP and their mixture were measured at room temperature. The fluorescence measurements were performed at different temperatures (298, 302, 306 and 310 K), and excitation wavelength was 280 nm. The excitation and emission slit widths were set at 2.5 nm. Appropriate blanks cor-responding to the buffer were subtracted to correct background fluorescence.

Results and discussion

Fluorescence spectrum

The residues of tryptophan and tyrosine on BSA have the fluorescent properties. With excitation wave-length of 280 nm, the emission wavelength is 331 nm, while YbTMPyP has no emission with the same excita-tion wavelength. Under this condition, the fluorescence spectra of BSA and YbTMPyP would not interfere with each other. The concentrations of BSA were stabilized at 10−5 mol•L−1, and the content of YbTMPyP was var-ied from 0 to 5×10−5 mol•L−1. The effect of YbTMPyP on BSA fluorescence intensity is shown in Figure 2.

Figure 2 The fluorescence spectrum of BSA in the presence of YbTMPyP. Concentration of YbTMPyP: 0, 0.8×10−5, 1.2×10−5, 1.6×10−5, 2×10−5, 3×10−5, 4×10−5, 5×10−5 mol•L−1 (from top to bottom).

Quenching mechanism

The intensity of fluorescence decreases by a wide variety of processes. Such decreases in intensity are called quenching. It is apparent from Figure 2 that the

fluorescence intensity of BSA decreased regularly with the increment of YbTMPyP concentration. The fluores-cence quenching data are usually analysed by the Stern- Volmer equation:12

F/F0＝1＋Kqτ0cq＝1＋KSVcq (1)

where F0 and F are the steady-state fluorescence inten-sities in the absence and presence of quencher- (YbTMPyP), respectively. KSV is the Stern-Volmer quenching constant, and cq the concentration of quencher. τ0 is fluorescent lifetime of BSA in the ab-sence of quencher. The Stern-Volmer quenching con-stant KSV of BSA and tryptophan residue fluorescence by YbTMPyP at different temperatures are shown in Table 1.

Table 1 Quenching constant KSV of BSA at different tempera-tures

Temperature/ K

Equation KS or KSV/

104 Kq/ 1012

298.15 (F0/F)－1＝1.96×104 cq 1.96 1.96

301.15 (F0/F)－1＝1.53×104 cq 1.53 1.53

304.15 (F0/F)－1＝1.14×104 cq 1.14 1.14

307.15 (F0/F)－1＝0.79×104 cq 0.97 0.97

310.15 (F0/F)－1＝0.38×104 cq 0.38 0.38

The fluorescence quenching includes both dynamic and static models. Under the model of static quenching, the Stern-Volmer quenching constant increased with the temperature increasing. The intensity of fluorescence of BSA was determined at different temperatures (298.15, 301.15, 304.15, 307.15, 310.15 K) in the presence of YbTMPyP, which is shown in Figure 3. According to values in Table 1, the quenching process of YbTMPyP is a kind of static quenching. Eq. (1) can be simplified as:13

F/F0＝1＋KScq (2)

where KS is formation constant of complex of BSA with YbTMPyP.

Figure 3 Relationship of [(F0/F)－1]-cq at different tempera-tures.

Porphyrin Chin. J. Chem., 2007 Vol. 25 No. 7 919

© 2007 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Thermodynamic parameters

According to Lineweaver-Burk equation for static quenching:14

1/(F0－F)＝1/F0＋1/KLBF0cq (3)

where KLB is the binding constant for static quenching, which can describe the equilibrium between BSA and YbTMPyP. According to data in Figure 3, KLB at dif-ferent temperatures can be obtained. Furthermore, if ∆H was considered as a constant value in a small range of temperature, several thermodynamic parameters could be obtained by the following equations.

1 2 2

2 1 1

lnRT T K

HT T K

∆ ＝

－

(4)

lnG RT K∆ ＝－ (5)

H GS

T

∆ ∆∆ －

＝ (6)

According to the values in Table 2, it can be seen that the binding process has the negative values of

r mH∆ � and r mS∆ � . The standard enthalpy r mH∆ � can be considered as an indicator of the increase of inter-molecular bond energies in binding process, while the standard entropy r mS∆ � reflect the decrease of disorder of the system during the reaction process. With the changes of enthalpy and entropy in this experiment having the opposite contribution, the binding of YbTMPyP and BSA is a “enthalpy-driven” reaction.15

Energy transfer between BSA and YbTMPyP

The Förster theory of molecular resonance energy transfer16 concluded that in addition to radiation and reabsorption, a transfer of energy could also take place through direct electrodynamic interaction between the primarily excited molecule and its neighbors.

According to this theory, the distance r could be calculated by the Eq. (7)17

60

6 60

RE

R r＝

＋ (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%.

6 25 2 40 8.9 10R K N JΦ－ －

＝ × (8)

where K2 is the orientation factor related to the geome-try of the donor and acceptor of dipoles and K2

＝2/3 for random orientation as in fluid solution, N is the average refracted index of medium in the wavelength range, Φ is the fluorescence quantum yield of the donor, and J is the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor.

According to the processing method reported in lit-eratures,8,13 the distance between BSA and YbTMPyP was obtained: r＝3.89 nm (Figure 4). After binding re-action, the YbTMPyP is so close to the residues of BSA that molecular resonance energy transfer occurs be-tween them.

Figure 4 Spectral overlap of YbTMPyP absorption and BSA fluorescence with the molar ratio of 1∶1.

Absorption spectrum of YbTMPyP

Figure 5 and shows the UV absorption spectra of YbTMPyP in the absence and presence of BSA. Addi-tion of BSA to YbTMPyP solution caused a small red drift (∆λ＝5 nm) and hypochromicity. These spectro-scopic variations indicate that the electron cloud of YbTMPyP has been dispersed. Porphyrin complexes have bound to BSA very closely, with the distance iden-tical to the obtained distance between BSA and YbTMPyP.

Table 2 Thermodynamic parameters of binding reaction

Temperature/K Linear equation KLB r mH∆ � /(kJ•mol－1) r mG∆ � /(J•mol－1) r mS∆ � /(J•mol－1•K－1)

298.15 (F0－F)－1＝0.0034＋4.69×10－7 1

qc－ 7242 －22.03 －10.77

301.15 (F0－F)－1＝0.0013＋1.97×10－7 1

qc－ 6545 －21.98 －10.83

304.15 (F0－F)－1＝0.0012＋2.18×10－7 1

qc－ 5904 －25.24 －21.96 －10.78

307.15 (F0－F)－1＝0.0015＋2.71×10－7 1

qc－ 5478 －21.99 －10.58

310.15 (F0－F)－1＝0.0014＋2.94×10－7 1

qc－ 4988 －21.96 －10.58

920 Chin. J. Chem., 2007, Vol. 25, No. 7 LIU et al.

© 2007 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 5 Absorption spectrum of YbTMPyP. a, without BSA and b, in the presence of BSA.

References

1 Lauceri, R.; Purrello, R.; Shetty, S. J.; Vicente, M. G. H. J. Am. Chem. Soc. 2001, 123, 5835.

2 Yin, Y. B.; Wang, Y. N.; Ma, J. B. Spectrochim. Acta Part A: Mol. Biomol. Spectros. 2006, 64, 1032.

3 Salvatore, S.; Antonino, M.; Luigi, M. S.; Francesca, M. M.; Vincenza, V.; Maria, T. S. Biomaterial 2006, 27, 4256.

4 Peng, X. B.; Liang, S. Q. Prog. Chem. 2001, 13, 360 (in

Chinese). 5 Wong, W. K.; Hou, A. X.; Guo, J. P.; He, H. S.; Zhang, L.

L.; Wong, W. Y.; Li, K. W.; Cheah, K. W.; Xue, F.; Tho-mas, C. W. M. J. Chem. Soc., Dalton Trans. 2001, 3092.

6 Masuoka, J.; Saltman, P. J. Biol. Chem. 1994, 269, 25557. 7 Sadler, P. J.; Viles, J. H. Inorg. Chem. 1996, 35, 4490. 8 Hu, Y. J.; Liu, Y.; Jiang, W.; Zhao, R. M.; Qu, S. S. J.

Photochem. Photobiol. B: Biol. 2005, 80, 235. 9 Hirayama, K.; Akashi, S.; Furuya, M.; Fukuhara, K. I. Bio-

chem. Biophys. Res. Commun. 1990, 173, 639. 10 Peters, T. Adv. Protein Chem. 1985, 37, 161. 11 Wong, W. K.; Zhang, L. L.; Wong, W. T.; Xue, F.; Thomas,

C. W. M. J. Chem. Soc., Dalton Trans. 1999, 615. 12 Lakowicz, J. R. Principles of Fluorescence Spectroscopy,

2nd Ed., Plenum Press, New York, 1999, p. 239. 13 Hu, Y. J.; Liu, Y.; Pi, Z. B.; Qu, S. S. Bioorg. Med. Chem.

2005, 13, 6609. 14 Ma, G. B.; Gao, F.; Ren, B. Z.; Yang, P. Acta Chim. Sinica

1995, 53, 1193 (in Chinese). 15 Paola, G.; Valeria, F.; Gastone, G. J. Phys. Chem. 1994, 98,

1515. 16 Förster, T. Ann. Phys. 1948, 2, 55. 17 Il’ichev, Y. V.; Perry, J. L.; Simon, J. D. J. Phys. Chem. B

2002, 106, 452.

(E0609184 LI, W. H.; DONG, H. Z.)