The Studies Of Protein By Spectral Methods

Protein is the most important life substance and plays an important role in animal life, such as poviding power, serving as the carrier of drug, microorganism and minerals. The amount of protein in human body is a reference for health. So, to quantify protein in biochemistry is very important, which attracts the attentions of many scientists. The scientists focus on finding a simple, quick and high sensitive method to determine the protein. There are more and more probes for determination of protein. To study the interaction between protein and probes become very important. In this thesis, the assay for protein and the interaction between protein and colloidal gold as well as Eosin Y were studied by spectral methods, including resonance light scattering (RLS) technique, fluorescence and absorption spectrometry.The surfactant always cooperates with RLS probe to enhance the RLS signal of system in the RLS technique for determination of proteins. In our knowledge, there were very few reports for the use of surfactant as a pobe to determine protein. In this thesis, the RLS spectra of sodium dodecylbenzene sulphonate (SDBS)-human complement factor C4,hyperin(Hy)-cetyltrimethylammonium bromide (CTMAB)-Fibronectin and CTMAB-SDBS-bovine serum albumin(BSA) system were investigated. The functions of surfactant in different system were discussed. Based the experimental results, the RLS mehods for determination of protein were provided. The effects of experimental parameters, including surfactant concentration, pH, incubation time and foreign substances, on the enhancement of RLS intensity were investigated. The assay for protein was successfully applied to determination of protein in synthetic samples.Colloidal gold has unique physical and chemical characteristics, and has been applied extensively in many areas. The interactions between colloidal gold and serum albumins, including BSA and human serum albumin (HSA), were studied by fluorescence and absorption spectrometry. The colloidal gold binding with serum albumins was determined to be static quenching process based on the determination of kq, the comparison of the values of KSV at different temperature and the comparison of absorption spectra of colloidal gold in the presence and absence of serum albumins. Based on the effect of colloidal gold on fluorescence intensity of serum albumins, the binding constants, the numbers of binding sites and the acting forces between colloidal gold and serum albumins were obtained. On the other hand, it was found that the interaction degree between colloidal gold and BSA was different with that between colloidal gold and HSA.The interaction between colloidal gold and protein (C4, BSA and HSA) at different pH values was investigated by spectral methods, including absorption and RLS spectrometry. Protonized protein can destroy the electrical double layer of colloidal gold and induce the aggregation of colloidal gold. According to the changes of color and absorption spectra of colloidal gold in the presence of protein, the interaction of colloidal gold and protein was studied and quantified using a"flocculation"parameter. The experimental results indicated that the aggregation of colloidal gold is relative to the structure, concentration and isoelectric point of protein. The results obtained from absorption spetra were similar to those obtained from TEM images and RLS spetra, which indicated that the absorption spectrometry can be successfully applied to the study of aggregation of colloidal gold. The Mey experimental results also proved the above conclusion to be true.The pH-dependent binding of Eosin Y and BSA was investigated by spectral methods, including RLS, absorption and fluorescence spectrometry. The experimental results showed that the fluorescence and RLS intensity of Eosin Y were dependent on the solution pH value, which indicated that the change of pH affected the structure of Eosin Y in solution. Based on the fluorescence data obtained, the style of binding, the binding constant, the binding site number and the other thermodynamic parameters for the interaction of BSA and Eosin Y were studied. Based on F?rester non-radiation energy transfer theory, the distance between donor BSA and acceptor Eosin Y was obtained. At different pH solution, conformational isomer of BSA and molecular structure Eosin Y were different, which may be responsible for the different binding constants for BSA and Eosin Y at different pH.