羟丙基-β-环糊精对1-羟基芘与牛血清白蛋白相互作用的影响

doi:10.7503/cjcu20160514

摘要/Abstract

摘要：

在模拟生理条件下, 利用荧光光谱、圆二色光谱和紫外-可见吸收光谱探究了羟丙基-β-环糊精(HPCD)对1-羟基芘(1-OHPyr)与牛血清白蛋白(BSA)相互作用的影响及其相关机制. 结果表明, 291 K下HPCD可使1-OHPyr与BSA的结合常数降低为1.45×10⁵ L/mol; 导致1-OHPyr-BSA体系中BSA的α-螺旋含量恢复, 色氨酸(TRP)残基周围微环境的极性变化减弱; 致使1-OHPyr及BSA的荧光寿命均延长, 且二者的结合距离增大. 初步研究结果表明, HPCD与1-OHPyr的包络作用是HPCD影响BSA与1-OHPyr结合的主要原因.

关键词: 1-羟基芘, 牛血清白蛋白, 羟丙基-β-环糊精, 光谱法, 影响机制

Abstract:

Fluorescence spectra, circular dichroism spectra and UV visible absorption spectra were employed to investigate the feasibility of regulating the interaction of 1-hydroxypyrene(1-OHPyr) with bovine serum albumin(BSA) using hydroxypropyl-β-cyclodextrin(HPCD) under simulated physiological conditions, and the related mechanism was preliminary discussed. The results showed that HPCD can inhibit the interaction of 1-OHPyr with BSA, and the binding constant of 1-OHPyr-BSA was decreased to 1.45×10⁵ L/mol at 291 K. HPCD can also restore the helix of BSA and inhibit the polarity changes of the local microenvironment of tryptophan(TRP) residues in BSA. HPCD can increase the fluorescence lifetimes of 1-OHPyr and BSA in 1-OHPyr-BSA system, meanwhile increase the binding distance of 1-OHPyr with BSA. The inclusion behavior of HPCD with 1-OHPyr was primarily the main reason why HPCD affected the binding of 1-OHPyr with BSA.

Key words: 1-Hydroxypyrene, Bovine serum albumin, Hydroxypropyl-β-cyclodextrin, Spectroscopic method, Influencing mechanism

中图分类号:

O657
O625

TrendMD:

张静, 陈霖锋, 朱亚先, 张勇. 羟丙基-β-环糊精对1-羟基芘与牛血清白蛋白相互作用的影响. 高等学校化学学报, 2017, 38(1): 28.

ZHANG Jing, CHEN Linfeng, ZHU Yaxian, ZHANG Yong. Effects of Hydroxypropyl-β-cyclodextrin(HPCD) on the Interaction of 1-Hydroxypyrene with Bovine Serum Albumin^†. Chem. J. Chinese Universities, 2017, 38(1): 28.

图/表 10

Fig.1 Fluorescence emission spectra of 1-OHPyr-BSA system in the presence of HPCD105 c(1-OHPyr)/(mol·L-1), a—f: 0, 0.2, 0.4, 0.5, 0.6, 0.8, 1.0; c(BSA)=1.0×10-6 mol/L; c(HPCD)=3.0×10-3 mol/L, 291 K. (A) Ex. slit: 1 nm; Em. slit: 1 nm; (B) Ex. slit: 2 nm; Em. slit: 2 nm.

Fig.2 Stern-Volmer plot(A) and double logarithm plot(B) for the quenching of BSA by 1-OHPyr in the presence of HPCD

Fig.3 Effects of HPCD on the synchronous fluorescence spectra of 1-OHPyr-BSA systemΔλ=60 nm; c(BSA)=5.0×10-6 mol/L; 105 c(1-OHPyr)/(mol·L-1): a. 0; b. 0.5; c. 1.0. c(HPCD)=3.0×10-3 mol/L.

Table 1 Effects of HPCD on the characteristics of BSA synchronous fluorescence peak in 1-OHPyr-BSA system*

c(1-OHPyr)/(mol·L^-1)	1-OHPyr-BSA		1-OHPyr-BSA-HPCD
c(1-OHPyr)/(mol·L^-1)	λ_max/nm	F/a.u.	λ_max/nm	F/a.u.
0	282	1.58×10⁶	282	1.54×10⁶
1.0×10^-6	284	1.15×10⁶	284	1.27×10⁶
5.0×10^-6	285	7.43×10⁵	285	9.27×10⁶
1.0×10^-5	286	4.32×10⁵	285	6.25×10⁶

Fig.4 CD spectra of the 1-OHPyr-BSA systems with different concentrations of HPCDc(BSA)=4.0×10-8 mol/L; c(1-OHPyr)=4.0×10-7 mol/L; c(HPCD): a. 3.0×10-5 mol/L; b. 3.0×10-4 mol/L.

Table 2 Secondary structural contents of BSA in different systems

System	α-Helix(%)	β-Sheet(%)	Turn(%)	Random coil(%)	NRMSD^a
BSA	50.9	11.6	13.1	24.5	0.101
1-OHPyr-BSA	54.5	8.2	14.6	22.7	0.083
1-OHPyr-BSA-HPCD^b	53.6	11.0	8.9	26.6	0.471
1-OHPyr-BSA-HPCD^c	51.8	10.2	12.0	26.1	0.094

Fig.5 Fluorescence decay curves for 1-OHPyr (A) and BSA (B) in different systems(A) λex=346 nm, λem=386 nm; (B) λex=282 nm, λem=341 nm; c(BSA)=c(1-OHPyr)=5.0×10-6 mol/L; c(HPCD)=3.0×10-3 mol/L.

Table 3 Fluorescence decay parameters for BSA and 1-OHPyr in different systems at 291 K*

System	τ_f(1-OHPyr)/ns	χ²	System	τ_f(BSA)/ns	χ²
1-OHPyr	14.73	1.066	BSA	5.98	1.051
1-OHPyr-HPCD	18.04	1.069	BSA-HPCD	6.06	1.157
1-OHPyr-BSA	16.21	1.070	1-OHPyr-BSA	4.59	1.024
1-OHPyr-BSA-HPCD	17.01	0.997	1-OHPyr-BSA-HPCD	5.07	1.117

Fig.6 Overlap of UV-Vis absorption spectrum of 1-OHPyr(a) in the presence of HPCD with the fluorescence emission spectrum of BSA(b)c(BSA)=c(1-OHPyr)=5.0×10-6 mol/L; c(HPCD)=3.0×10-3 mol/L.

Table 4 Energy transfer parameters between 1-OHPyr and BSA in the absence and presence of HPCD*

System	J/(cm³·L·mol^-1)	E(%)	R₀/nm	r/nm
1-OHPyr-BSA^[6]	1.37×10^-14	40	2.69	2.88
1-OHPyr-BSA-HPCD	2.16×10^-14	21	2.85	3.55

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