Preliminary Study On The Phosphoamino Acids And Phosphoprotein By Fluorescence Probe

Protein phosphorylation/dephosphorylation plays a central role in biological systems. Often, phosphorylation/dephosphorylation acts as a molecular switch controlling the protein activity in different pathways as in cell proliferation, differentiation metabolism, and signal transduction etc. The typical acceptors for eukaryotic phosphorylation are serine (Ser), threonine (Thr) and tyrosine (Tyr). The phosphorylation/dephosphorylation of these amino acids is a topic of current interests. Many groups have been focused their attention on this field. The detection of phosphorylated amino acids in the presence of amino acids is important for understanding the role of many proteins. The Above consideration invited us to develop a highly sensitive and selective method for the detection of phosphorylated amino acids. In the present dissertation, the Tb³⁺-Tiron complex, a fluorescence probe, is successfully proposed to investigate the phosphoamino acid and phosphoprotein. This dissertation consists of four chapters.In chapter 1, the research progresses in phosphoamino acids and phosphoprotein is briefly reviewed, including the detection, identification and quantitative analysis of phosphoamino acids and phosphoprotein. Both the luminescence mechanism and applications of the rare-earth ion complex are also discussed here. Based on the research progresses in this area and previous works in our own lab, the research proposals of this dissertation are proposed in this chapter.In chapter 2. Previous works in our own lab have found that some compounds with phosphate groups, such as polynucleotides, nucleic acids, lecithin, coenzymes and conjugated proteases, can effectively quench the fluorescence of Tb³⁺-Tiron complex. Thus, the possibility of investigating phosphoamino acids with Tb³⁺-Tiron complex was explored in this chapter, and a possible mechanism of the interaction of the Tb³⁺-Tiron complex with various compounds of phosphate groups was also proposed. The feasibility of using the Tb³⁺-Tiron complex as a fluorescence probe to monitor the hydrolysis process of phosphoamino acids was also analysed in this chapter. In chapter 3. Based on the result obtained in chapter 2, in this chapter, Tb³⁺-Tiron complex fluorescence probe was used to monitor the hydrolysis process of phospho-Ser, phospho-Thr and phospho-Tyr. The quenching of the fluorescence emission of Tb³⁺-Tiron complex is concentration dependent. A new method, therefore, was developed for the sensitive and selective determination of phosphoamino acids. The linear range of P-Ser, P-Thr and P-Tyr are 5.0×10-8 ⁵.0×10-7 mol/L , 5.0×10-8 ⁶.0×10-7 mol/L and 5.0×10-8 ⁶.0×10-7 mol/L, respectively; the limit of detection is 2.06×10-8 mol/L, 1.13×10-8 mol/L and 1.74×10-9 mol/L, respectively.In the last chapter. Since various amino acids do not interfere the fluorescence quenching of Tb³⁺-Tiron complex, in this chapter, such fluorescence probe was used to detect the content of phosphate groups in phosphoprotein. Phosvitin, a P-Ser containing protein, was used as a model protein to confirm the present method. The content of phosphate groups in phosphoprotein obtained by the present method is consistent with that reported by the manufacturer. The number of P-Ser residue in each phosvitin calculated from the obtained data is 98. The confirmation of the present method provides a possibility for further investigating other phosphoprotein.