[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 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%.
Lin
Xu-Cong
XC
Chemistry and Engineering College of Fuzhou University, Fuzhou 350002, China. xulin@fzu.edu.cn
Yan
Jin
J
Guo
Liang-Qia
LQ
Xie
Zeng-Hong
ZH
D000818:Animals; D002232:Carbocyanines / Q000737:chemistry*; D002417:Cattle; D003300:Copper / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D024841:Fluoroquinolones / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000737:chemistry; D015335:Molecular Probes / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000737:chemistry
D002232:Carbocyanines; D005456:Fluorescent Dyes; D024841:Fluoroquinolones; D015335:Molecular Probes; D012709:Serum Albumin; C547576:sinafloxacin; D003300:Copper; D008274:Magnesium; D015032:Zinc