吲哚基二聚体菁类探针同步荧光测定蛋白质的研究

基于蛋白质对双嵌吲哚染料具有良好的荧光增强作用，以新型水溶性吲哚基同型二聚体探针I，建立了一种灵敏的蛋白质同步荧光分析体系。实验考察了吲哚探针的荧光特征、吲哚探针浓度、缓冲体系pH、盐浓度等参数对体系荧光的影响。在酸性条件下，蛋白质分子与探针I发生结合作用，同步荧光明显增强并向长波方向发生红移，且同步荧光强度与蛋白质浓度成良好的线性关系。在最优条件下，牛血清白蛋白BSA的线性响应范围5.00×10-7～2.50×10-5 g·mL-1,检测限(3σ/K)为3 ×10-8 g·mL-1；测定了血清蛋白BSA的合成样品，不同浓度BSA样品回收率为98.6%～103.0%，相对标准偏差1.1%～1.9%；与蛋白质紫外标准测定法比较，测定偏差为0.4%～3.9％。

Determination of Protein by Synchronous Fluorometric Method with a New Indole Homodimeric Cyanine as Fluorescence Probe

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 α-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 (ΔF) gained the peak at 3.00 μmol·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 5.00×10-7-2.50×10-5 g·mL-1 (BSA) with a detection limit of 3×10-8 g·mL-1. Applied to simulant protein samples at the level of 1.00, 2.00, and 5.00 μg·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%.