Novel Quantitative Analysis Methods For Proteins And Research Of The Interaction Of Proteins With Small Molecules

Protein is one of the most important substances investigated in life sciences. Human Serum Albumin (HSA) is the most abundant carrier protein in blood circulation. It has many important physiological and pharmacological functions, which can bind many exogenous and endogenous ligands in blood, and realize transport and distribution of many molecules and metabolites, such as fatty acids, amino acids, hormones, ions and many diverse drugs. Human Immunoglobulin (γ-G), as cell surface receptor of lymphocyte, plays important roles in many cell actions, it also plays a key role in human immune response. Therefore, HSA andγ-G have been the most extensively studied proteins. Investigating novel quantification method of proteins with high sensitivity and selectivity and researching the binding mechanism and process of toxic materials and drugs with proteins have many importances in life sciences, clinical medicine, toxicology and pharmacokinetics. Thus, it has been an interesting research field of life sciences, chemistry and clinical medicine. In this dissertation, on the basis of the previous research, the resonance light scattering technique, fluorescence spectroscopy combined with UV-visible absorption, Fourier transform infrared (FT-IR), circular dichroism (CD) spectroscopy along with computational modeling were used to investigate the novel quantification method of proteins and research the interaction of several organic small molecule substances with proteins. The following major innovative works were carried out:1. The total internal reflection and flow injection combined with resonance light scattering technique were used to quantify the protein in human serum samples. The homemade instruments of total internal reflection and flow injection were connected with spectrofluorophotometer. Protein in human serum samples were determined using the combination techniques mentioned above, the results were consistent with that ones obtained by the first Hospital of Lanzhou University.2. The interaction and binding mechanism of Arsenazo-TB, Cal-Red and Daidzein withproteins were investigated, the binding parameters including binding constants and the number of binding sites were calculated according to Scatchard and Stern-Volmer equation, the thermodynamic parameters were also calculated according to van't Hoff equation and the main interaction force between organic small molecules and proteins was discussed.3. The alternations of proteins' secondary structure caused by the binding of smallligands were estimated by Fourier transform infrared (FT-IR), circular dichroism (CD) spectroscopic methods. The computational modeling method was used to study the dye-HSA interaction, the molecular model of the interaction of dye and HSA was obtained and the results were consistent with the experimental results.4. The interaction of Daidzein withγ-G in water/oil microemulsions has been investigated and compared with that of in aqueous solution, the influence of the size of water pool in microemulsions on the interaction of Daidzein withγ-G was also investigated by fluorescence polarization and dynamic laser scattering technique.This dissertation consists of seven chapters:Chapter 1: The structures, functions and natures of proteins were briefly described. The methods of determination of proteins and the study of interaction of organic small ligands with proteins were reviewed.Chapter 2: A novel quantification method of protein in human blood serum samples was proposed based on the measurement of total internal reflected resonance light scattering (TIR-RLS) at water/tetrachloromethane (H₂O/CCl₄) interfaces. At pH 4.10 and in the presence of Cetyltrimethylammonium bromide (CTAB), the interaction of Arsenazo-TB (ATB) with human serum albumin (HSA) results in strong enhanced TIR-RLS signals with the maximum spectra peak located at 330 nm. The enhanced TIR-RLS intensity was found to be proportional to the concentration of HSA in the range of 0.5-42.3μg/mL and its limit of determination (3σ) is 198 ng/mL. The content of HSA in six blood serum samples was determined with the maximum RSD of 1.2% and the recovery of 97-103%.Chapter 3: A novel method of flow injection sampling combined with resonance light scattering detection was developed for the determination of total protein. This method is based on the enhancement of RLS signals from methyl blue (MB) by protein at pH 4.1. The enhanced RLS intensities at 333 nm were proportional to the protein concentration over the range of 2.0-37.3μg/mL and 1.0-36.0μg/mL for human serum albumin (HSA) and bovine serum albumin (BSA), respectively. The corresponding limits of detection (3a) of 45 ng/mL for HSA and 80 ng/mL for BSA were attained. The method was successfully applied to the quantification of total proteins in five human serum samples, the maximum relative error was less than 1% and the recovery was between 98%-102%, The sample throughput was 60 h^-1.Chapter 4: A novel method of flow injection sampling combined with resonance light scattering detection was developed for the determination of protein concentration in human serum samples. This method is based on the enhanced RLS signals of proteins binding with acid chrome blue K (ACBK) at acidic aqueous solution of pH 4.10. The enhanced RLS intensities at 264 nm were proportional to the proteins concentration over the range of 2.0-40.0μg/mL and 4.0-33.8μg/mL for human serum albumin (HSA) and bovine serum albumin (BSA), respectively. The corresponding limits of detection (3σ) of 85 ng/mL for HSA and 124 ng/mL for BSA were attained. This method was successfully applied to the quantification of total proteins in six human serum samples, the maximum relative standard deviation was less than 2% and the recovery was between 97%-103% for standard addition method, the sample throughput was 60 h^-1.Chapter 5: The interaction between Arsenazo-TB and human serum albumin (HSA) was studied by Resonance light scattering (RLS), Fourier transformed IR (FT-IR) and circular dichroism (CD) spectroscopic methods. The binding parameters of Arsenazo-TB with HSA were studied at different temperatures under the optimum conditions. It is indicated that the binding parameters decreased with the increasing of the temperature. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy was hydrophobic interaction. The alterations of protein secondary structure in the presence of Arsenazo-TB were quantitatively calculated with reductions ofα-helix and with increases ofβstructures.Chapter 6: The interaction between Cal-Red and human serum albumin (HSA) was studied using Resonance light scattering (RLS), Fourier transformed Infrared (FT-IR), Circular dichroism (CD) and molecular modeling methods. The binding parameters of HSA with Cal-Red were studied at different temperatures and it is indicated that the binding constant K and the maximum binding number N decreased with increasing the temperature. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy was hydrophobic interaction. The alterations of protein secondary structure in the presence of Cal-Red were quantitatively calculated with reductions ofα-helix andβ-turn structures and with increases ofβ-sheet structure. The primary binding site of Cal-Red was located in IIA subdomain of HSA.Chapter 7: The interaction of Daidzein withγ-G in reverse microemulsions of H₂O/AOT/isooctane at simulative physiological pH has been investigated by fluorescence, UV-absorption, Circular dichroism (CD) spectroscopic methods and dynamic laser scattering technique. The results of spectroscopic measurements suggested that the intrinsic fluorescence ofγ-G was quenched by Daidzein through static quenching mechanism and the interaction was via a single class of binding site. The binding constant K decreased with increasing the temperature and the binding force in emulsion was larger than that of in aqueous solution. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy was hydrophobic interaction. The result of CD spectroscopy suggested that the secondary structure ofγ-G changed after interacted with Daidzein.