[以新型吲哚同二聚体花菁为荧光探针的同步荧光法测定蛋白质]

[Determination of protein by synchronous fluorometric method with a new indole homodimeric cyanine as fluorescence probe].

作者信息

Lin Xu-Cong, Yan Jin, Guo Liang-Qia, Xie Zeng-Hong

机构信息

Chemistry and Engineering College of Fuzhou University, Fuzhou 350002, China.

出版信息

Guang Pu Xue Yu Guang Pu Fen Xi. 2008 Nov;28(11):2615-8.

PMID:19271502

Abstract

Using a new homodimeric hydrophilic indole dye (Dye-I) as fluorescence probe, a sensitive synchronous spectrofluorometric determination for protein was developed. Characteristics of the fluorescence reaction between DYE-1 and BSA protein were investigated. Effects of the concentration of the hydrophilic dye, pH value of the buffer solution, and ion-intensity of NaCl were also studied and the optimum condition was gained. At pH of 2.50, electrostatic interactions of positive charges of the BSA chain and negative charges on the sulfonic groups of DYE-1 were carried out. The interactions of the indole group of DYE-1 and some active groups of BSA (viz. amido, carboxyl or sulfhydryl) were also achieved, and resulted in the combination of indole group of dye at the chain of BSA, which caused a notable increase in synchronous fluorescence with an observable shift to the longer emission wavelength. Effects of the concentration of indole dye on the determination of BSA were also investigated. With the augmentation of BSA, the alpha-helix structure of BSA molecular would change from the unwrapped state to the enfolded state, which was in favor of restraining free-oscillation of fluorescence probe in the solution and maintaining a high energy transfer efficiency. Such a fact would fuel a high fluorescence enhancement and the change in fluorescence intensity (deltaF) gained the peak at 3.00 micromol x L(-1). The influences of ion-intensity of NaCl on the fluorescence of BSA-DYE-1 system was visible. Effects of coexistent substances such as amino acid and metal ions such as Cu2+, K+, Ca2+, Mg2+, Al3+, and Zn2+ were also investigated. Most substances showed no notable influences on the determination of BSA except Zn2+ and Cu2+ ions. Under the optimum conditions, good calibration curves of the protein were also obtained in the range of 5.00 x 10(-7) -2. 50 x 10(-5) g x mL(-1) (BSA) with a detection limit of 3 x 10(-8) g x mL(-1). Applied to simulant protein samples at the level of 1.00, 2.00, and 5.00 microg x mL(-1) of BSA, the recoveries were in the range of 98.6%-103.0% with the RSD of 1.1%-1.9%. Compared with the UV standard method, the relative deviation was obtained in the range of 0.4%-3.9%.

摘要

以一种新型同二聚体亲水性吲哚染料（染料-I）为荧光探针，建立了一种灵敏的蛋白质同步荧光光度测定法。研究了DYE-1与牛血清白蛋白（BSA）蛋白之间荧光反应的特性。考察了亲水性染料浓度、缓冲溶液pH值和NaCl离子强度的影响，获得了最佳条件。在pH为2.50时，BSA链上的正电荷与DYE-1磺酸基团上的负电荷发生静电相互作用。DYE-1的吲哚基团与BSA的一些活性基团（即酰胺基、羧基或巯基）也发生了相互作用，导致染料的吲哚基团在BSA链上结合，使同步荧光显著增强，发射波长明显向长波方向移动。还考察了吲哚染料浓度对BSA测定的影响。随着BSA的增加，BSA分子的α-螺旋结构将从展开状态转变为折叠状态，这有利于抑制荧光探针在溶液中的自由振荡并保持较高的能量转移效率。这一事实将导致荧光大幅增强，荧光强度变化（ΔF）在3.00 μmol·L⁻¹时达到峰值。可见NaCl离子强度对BSA-DYE-1体系荧光有影响。还考察了氨基酸等共存物质以及Cu²⁺、K⁺、Ca²⁺、Mg²⁺、Al³⁺和Zn²⁺等金属离子的影响。除Zn²⁺和Cu²⁺离子外，大多数物质对BSA的测定无显著影响。在最佳条件下，在5.00×10⁻⁷ - 2.50×10⁻⁵ g·mL⁻¹（BSA）范围内也获得了良好的蛋白质校准曲线，检测限为3×10⁻⁸ g·mL⁻¹。应用于BSA水平为1.00、2.00和5.00 μg·mL⁻¹的模拟蛋白质样品，回收率在98.6% - 103.0%范围内，相对标准偏差为1.1% - 1.9%。与紫外标准方法相比，相对偏差在0.4% - 3.9%范围内。