Studies On The Analysis Of Proteins And Nucleic Acids By Resonance Light Scattering Technique And Their Applications

The Resonance Light Scattering (RLS) technique was set up by Pastemack and co-workers using common fluorimetric instrument in 1993. They studied distinguishing, assembly, and aggregation on bimolecular by RLS technique. People follow the RLS with great interest.Protein and nucleic acid are fundamental materials of life. They plays a very important role in process of life. With the effort of a great deal of scientists, people have made much breakthrough in the studies on protein and nucleic acid during the last century. Today, the study field of protein and nucleic acid has been extended. In the chemical studies on protein and nucleic acid, the field of small molecular is always very brighten, because it can in favor of exploiting new medicals of protein and nucleic acid. Armed with the advanced equipment and sensitive determination methods, analytical chemistry plays an important role in the studies on the interaction between biopolymer and small molecular for the quantitative analysis of biopolymer, and for the investigation of the mechanism.Based on a lot of documents, this thesis chooses several kinds of organic or drug small molecular, using resonance light-scattering technique, UV/Vis absorption spectra method, fluorescence spectra method and 1H NMR spectra method studied the interaction mechanisms of those small molecular and biopolymer. Base on the variation of the intensity of resonance light scattering is ratio to the concentration of the protein and DNA establishes several new determination methods of protein and DNA. These methods are simple and rapid with excellent reappearance, selectivity, sensitivity and wide linear coverage.This paper has five chapters.In the first chapter, we summarized the development of proteins and nucleic acids, the theory of RLS and the foundation of quantitative analysis, the application of RLS in the proteins and nucleic acids investigation and the analysis.In the second chapter , we studied the resonance light scattering spectral characteristic of tichromine-protein system, and the elementary reaction mechanism with absorption spectra method and a lot of condition experiments. Another new determination method of protein at nanogram level was established. In the Walpole (NaAc-HCl) buffer of pH 4.05, RLS of the tichromine-protein system with the maximum scattering peak located at 399 nm. Under the optimum conditions, the enhanced RLS intensity was proportional to the concentration of proteins, over the range of 0.0-0.2mg/L and 0.2-8.0 mg/L for BSA and 0.0-0.4 mg/L and 0.4-5.0 mg/L for HSA respectively, with a correlation coefficient of 0.9992, 0.9996 for BSA and 0.9995, 0.9993 for HSA, with the limits of determination of 8.3ng/mL for BSA and 7.4ng/mL for HSA. This method is simple, rapid and has been applied satisfactorily to determination of total proteins in human serum samples with satisfactory results.In the third chapter , we studied the resonance light scattering spectral characteristic of eriochrome red B-Sodium dodecyl sulphonate-protein system, and the elementary reaction mechanism with absorption spectra method and a lot of condition experiments. Another new determination method of protein at nanogram level was established. In the Brittion-Robinson buffer of pH 2.87, RLS of the eriochrome red B-Sodium dodecyl sulphonate-protein system with the maximum scattering peak located at 563 nm. Under the optimum conditions, the enhanced RLS intensity was proportional to the concentration of proteins, over the range of 0.0-2.0mg/L and 2.0-8.0 mg/L for HSA and 0.0-3.5 mg/L and 3.5-12.5mg/L for BSA respectively, with a correlation coefficient of 0.9997, 0.9996 for HSA and 0.9992, 0.9998for BSA, with the limits of determination of 22.6ng/mL for BSA and 12.0ng/mL for HSA. This method is simple, rapid and has been applied satisfactorily to determination of total proteins in human serum and creamery samples with satisfactory results.In the fourth chapter , we studied the resonance light scattering spectral characteristic of patent blueⅤ- Cetyltrimethyl ammonium bromide-nucleic acid system, and the elementary reaction mechanism with absorption spectra method and 1H NMR spectra method. Another new determination method of nucleic acid at nanogram level was established. In the Brittion-Robinson buffer of pH 9.9, RLS of the patent blueⅤ- Cetyltrimethyl ammonium bromide-nucleic acid system with the maximum scattering peak located at 345nm. Under the optimum conditions, the enhanced RLS intensity was proportional to the concentration of nucleic acids, over the range of 0.0-1.5 mg/L for yDNA, 0.0-1.6 for ctDNA and 0.0~2.0 for fsDNA, with a correlation coefficient of 0.9991, 0.9992 and 0.9989 respectively;with the limits of determination of 14.6ng/mL for yDNA, 15.4 ng/mL for ctDNA and10.2 ng/mL for fsDNA. This method is simple, rapid and has been applied satisfactorily to determination of DNA in synthetic samples.In the fifth chapter, the interaction between the antitumor drug tamoxifen and calf thymus DNA was studied by different spectroscopic methods including: UV/Vis absorption spectra, fluorescence spectra, resonmance light scattering technique and 1H NMR spectra. All the phenomenon of experiments indicated tamoxifen bind to ctDNA mainly by an intercalative force as well as electrostatic force. This conclusion was reinforced by salt effect and thermo-denaturation experiments. Intrinsic binding constant was 8.15×104 L/mol obtained by Stern-Volmer equation and the binding site size was about 2.8 obtained by Scatchard equation.