含磷三足体稀土铕(Ⅲ)配合物与牛血清白蛋白的作用机理

doi:10.7503/cjcu20141088

摘要/Abstract

摘要：

在pH=7.3的Tris-HCl缓冲溶液(模拟生理条件)中, 采用荧光光谱、循环伏安曲线和紫外光谱研究了N-二(苯-二氨基甲酰基)甲基磷酸铕(Ⅲ)配合物[Eu(pic)₃L]与牛血清白蛋白(BSA)的相互作用. 实验结果表明: 配合物与BSA可以形成1∶1结合型无荧光复合物Eu(pic)₃L-BSA, Eu(pic)₃L对 BSA 内源荧光的猝灭类型为静态猝灭. 根据双对数回归方程计算出二者在不同温度下的结合常数K及结合位点数n, 通过热力学参数得出配合物与 BSA 之间以氢键和范德华力为主. 根据Foster的偶极-偶极无辐射能量转移机理可知配合物与BSA之间可能以偶极-偶极无辐射能量转移方式进行能量传递. 分别考察了Fe³⁺和Cu²⁺对配合物与BSA结合作用的影响, 推测Fe³⁺和Cu²⁺可能在配合物与BSA间起“离子架桥”作用, 使Eu(pic)₃L-BSA复合物的稳定性增强. 循环伏安法研究结果表明配合物与BSA相互作用形成无电活性的Eu(pic)₃L-BSA复合物, 使得溶液中游离的配合物浓度降低.

关键词: N-二(苯-二氨基甲酰基)甲基磷酸, 铕(Ⅲ)配合物, 牛血清白蛋白, 作用机理

Abstract:

Under the simulated physiological condition(Tris-HCl buffer, pH=7.3), the interaction of N-bis(benzene-diamino formyl)methyl phosphate europium(Ⅲ) complex[Eu(pic)₃L] and bovine serum albumin(BSA) was studied by fluorescence spectrometry, cyclic voltammetry and UV spectroscopy. The results indicate that no fluorescent compound Eu(pic)₃L-BSA(ratio 1∶1) is formed and the complex can markedly quench the intrinsic fluorescence of BSA via a static quenching process. The binding constant(K) and binding site number(n) were calculated according to the double logarithm regression equation under different temperatures, the thermodynamic parameters show that the main force between complex and BSA is hydrogen bonding and van der Waals forces. The complex and BSA may have dipole-dipole non-radiation energy transfer accor-ding to the Foster’s theory of dipole-dipole non-radiation energy transfer. The effects of metal ions Fe³⁺and Cu²⁺on the interactions between BSA and complex were investigated, and the results show that Fe³⁺and Cu²⁺ may play “ion bridge” role in complex and BSA, making the stability of Eu(pic)₃L-BSA enhanced. Cyclic voltammetry results indicate that non-electric activity compound Eu(pic)₃L-BSA is formed, which lower the free complex concentration in the solution.

Key words: N-Bis(benzene-2 amino formyl)methyl phosphate, Europium(Ⅲ) complex, Bovine serum albumin(BSA), Interaction mechanism

中图分类号:

TrendMD:

杨水兰, 宋盼, 佘文洁, 杨天林. 含磷三足体稀土铕(Ⅲ)配合物与牛血清白蛋白的作用机理. 高等学校化学学报, 2015, 36(7): 1254.

YANG Shuilan, SONG Pan, SHE Wenjie, YANG Tianlin. Mechanism of the Interaction Between a Phosphorus-containing Tripod Ligand Europium(Ⅲ) Complex and Bovine Serum Albumin^†. Chem. J. Chinese Universities, 2015, 36(7): 1254.

图/表 17

Fig.1 Structure of Eu(pic)3L

Fig.2 Fluorescence emission spectra of BSA influenced by different concentrations of Eu(pic)3LT=303 K, pH=7.3, λex=280 nm, cBSA=1×10-7 mol/L. 107cEu(pic)3L/(mol·L-1) from a to i: 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0.

Fig.3 Stern-Volmer curves of BSA quenched by Eu(pic)3L at different temperatures

Table 1 KSV and kq data of Eu(pic)3L complex-BSA system at different temperatures

T/K	10^-5K_SV/(L·mol^-1)	10^-13K_q/(L·mol^-1·s^-1)	R²
293	4.16	4.16	0.996
303	3.88	3.88	0.993
313	3.15	3.15	0.995
323	3.10	3.10	0.996

Fig.4 Double logarithm regression curves of lg(F0/F-1) and lgcQ of Eu(pic)3L complex-BSA system at different temperatures

Table 2 Binding constants(K), binding sites(n) and R2 of Eu(pic)3L complex-BSA at different temperatures

T/K	Equation	K/(L·mol^-1)	R²	n
293	lg(F₀/F-1)=6.57+1.33lgc_Q	1.24×10⁶	0.991	1.33
303	lg(F₀/F-1)=6.39 +1.30lgc_Q	2.88×10⁵	0.993	1.30
313	lg(F₀/F-1)=6.14+1.26lgc_Q	1.51×10⁵	0.994	1.26
323	lg(F₀/F-1)=5.82+1.21lgc_Q	4.11×10⁴	0.994	1.21

Fig.5 Structure of BSA showing Trp-212 and Trp-134

Fig.6 Model of complex embedded to the hydrophobic pocket of BSA by hydrogen bonding

Table 3 Thermodynamic parameters of complex-BSA binding procedure

T/K	ΔH/(kJ·mol^-1)	ΔS/(J·K^-1)	ΔG/(kJ·mol^-1)
293	-107.76	-251.13	-34.19
303	-107.76	-251.12	-31.67
313	-107.76	-245.14	-31.03
323	-107.76	-245.29	-28.53

Fig.7 UV absorption spectrum of Eu(pic)3L complex(a) and fluorescence emission spectrum of BSA(b)

Fig.8 Quenching effect of Eu(pic)3L complex on BSA fluorescence in the presence of Cu2+(A) and Fe3+(B) T=303 K, pH=7.3, λex=280 nm, cCu2+-BSA or cFe3+-BSA=1×10-6 mol/L. 107cEu(pic)3L/(mol·L-1): a. 0; b. 1.0; c. 2.0; d. 3.0; e. 4.0; f. 5.0; g. 6.0; h. 7.0; i. 8.0; j. 9.0.

Fig.9 Stern-Volmer curves(A) and double logarithm plots(B) showing the Eu(pic)3L complex quenching effect on BSA fluorescence in the presence of Fe3+ and Cu2+ ions respectively(B)

Table 4 Binding parameters of Eu(pic)3L complex-BSA system in the presence of Fe3+ and Cu2+

System	K/(L·mol^-1)	n	R²
Complex-BSA	2.88×10⁵	1.30	0.993
Complex-BSA-Cu²⁺	9.98×10⁶	1.31	0.992
Complex-BSA-Fe³⁺	1.79×10⁶	1.38	0.998

Fig.10 Effect of Fe3+ and Cu2+ on UV spectra of Eu(pic)3L-BSAa. Eu(pic)3L-BSA; b. Eu(pic)3L-BSA-Fe3+; c. Eu(pic)3L-BSA-Cu2+.

Fig.11 CV curves of Eu(pic)3L complex with DNA(A) and the plot of 1/(1-i/i0) vs 1/cBSA(B)cEu(pic)3L=1×10-5 mol/L. cBSA/(10-6 mol·L-1) form a to f: 0; 1.0; 2.0; 3.0; 4.0; 5.0.

Fig.12 UV absorption spectra of Eu(pic)3L in the absence and presence of BSA(A) and the double

Table 5 Kb of Eu(pic)3L complex and BSA at different temperatures

System	T/K	10^-5K_b/(L·mol^-1)	R²
Complex-BSA	293	1.57	0.996
Complex-BSA	303	3.28	0.998

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