Detecttion Of Fructose Bisphosphate And Separation And Enrichment Of Phosphorylated Protein Based On Uranyl Complexes

In the first part of this paper, firstly the properties and detection methods offructose bisphosphate are introduced. Then the significance of proteinphosphorylation is introduced and the research development in the separation anddetermination of phosphorylated protein is summatezed. Meanwhile, the structurecharacteristics and applications of complexes of uranium with salophen are outlined.The properties and applications of Aminofluorescein are also introduced.In the second part, we report the synthesis and spectral properties of a conjugatesalophen-fluorescein and its uranyl complex designed to act as a fluorescence probesfor uranyl. The salophen containing carboxyl group is a tetradentate Schiff baseligand prepared by the reaction of salicylaldehyde with3,4-diaminobenzoic acid.Salophen-fluorescein was synthesized by connecting5-aminofluorescein (5-AF) tothe salophen through the condensation reaction of amino group with carboxyl group.The complex uranyl-salophen-fluorescein was obtained by the coordination reactionof salophen-fluorescein with uranyl cation. The spectral properties ofsalophen-fluorescein and its uranyl complex were studied by infrared spectrometry,ultraviolet visible absorption spectroscopy and fluorescence spectroscopy. Thefluorescence intensity of salophen-fluorescein is much stronger than that of5-AF, andmuch weaker than that of its uranyl complex, and the fluorescence intensity ofsalophen-fluorescein linearly increases with the increase of uranyl concentration.These facts demonstrate that salophen-fluorescein is a potential fluorescence probe foruranyl.In the third part, we report a double–receptor sandwich type fluorescence sensingmethod for the determination of fructose bisphosphates (FBPs) using fructose 1,6-bisphosphate (F-1,6-BP) as a model analyte based on uranyl-salophen complexes.The solid phase receptor is an immobilized uranyl-salophen (IUS) complex which isbound on the surface of glass slides by covalent bonds. The labeled receptor isanother uranyl-salophen complex containing a fluorescence group, oruranyl-salophen-fluorescein (USF). In the procedure of determining F-1,6-BP insample solution, F-1,6-BP is first adsorbed on the surface of the glass slide throughthe coordination reaction of F-1,6-BP with IUS. It then binds USF through anothercoordination reaction to form a sandwich-type structure of IUS-F-1,6-BP-USF. Theamount of F-1,6-BP is detected by the determination of the fluorescence intensity ofIUS-F-1,6-BP-USF bound on the glass slide. Under optimal conditions, the linearrange for the detection of F-1,6-BP is0.05to5.0nmol/mL with a detection limit of0.022nmol/mL. The proposed method has been successfully applied for thedetermination of F-1,6-BP in real samples with satisfactory results.In the fourth chapter, we introduce an immobilized metal ion chromatographymethod for the separation and enrichment of phosphorylated proteins. The method isbased on the specific adsorption of uranyl salophen to phosphate group. Wesynthesized a type of silica gel particles in which surface uranyl-salophen complex isbinded, or uranyl-salophen-silica gel (USSG) particles, and used the particles as thesolid phase receptor of phosphorylated proteins. Immobilized metal ionchromatography column was prepared with the USSG particles. In a separationprocedure, firstly proteins were converted by enzyme to form peptides. Then thepeptides were passed through the chromatography column. In the column thephosphorylated pepetides were captured by USSG particles. The capturedphosphorylated pepetides were then eluted by0.5mol/L hydrochloric acid. Thus thepurpose of separation and enrichment of phosphorylated proteins was achieved. Theexperimental results show that phosphorylated proteins can be effectively separatedand enriched by this method.