Study On The Interactions Between Drugs And BSA And On The Development Of Analytical Method For Protein Analysis

Serum albumin, the richest protein in plasma, can serve as a deposition and transportprotein for numerous endogenous and exogenous compounds. It is of great significance toinvestigate the interactions between serum albumin and drugs, since it can not only helpunderstand the transportation and distribution of drugs but also elucidate the mechanism,pharmacokinetics and toxicity of drugs. The technology of fluorescence is the mainmethod for researching the interactions between biomacromolecule and drugs.Proteins are the important element for vital movement and human health. The contentof protein affects the nutrition and quality of dairy products, so it has been a hot topic tostudy the analysis of proteins in dairy products. Capillary electrophoresis (CE) isincreasingly recognized as a highly attractive separation technique because of its highseparation efficiencies, short analysis time, small sample requirements and low operationcost. CE is widely used for analysis proteins. This thesis is mainly concerned with thefollowing aspects:(1) The investigation of the interactions between strychnine and bovine serumalbumin (BSA) was made by using fluorescence spectroscopy and ultraviolet spectroscopy.The static quenching and the non-radiation energy transfer were found to be the two mainreasons to leading the fluorescence quenching of BSA. The apparent combining constants(K_A) between strychnine and BSA are 3.72×10~3 at 27℃, 4.27×10~4at 37℃, 4.47×10~3at 47℃and the combining sites are 1.01±0.03. The combining distance (r=3.795 nm)and energy transfer efficiency (E=0.0338) are obtained by Forster's non-radiation energytransfer mechanism. The effect of strychnine on the structural changes of BSA was alsostudied using synchronous fluorescence spectroscopy. The experiment showed that theinteraction between strychnine and BSA was driven mainly by hydrophobic force. Theprocess of binding was a spontaneous.(2) A CE-based noncompetitive immunoassay for immunoglobulin G (IgG) in bovinecolostrum products was established. Fluorescein isothiocyanate labeled protein G(FITC-PrG) was tagged through non-covalent bindings to the Fc region of the mousemonoclonal anti-bovine IgG (Ab). The FITC-PrG, Ab and IgG formed a sandwiched immunocomplex FITC-PrG-Ab-IgG under optimal incubation conditions. Theimmunocomplex was separated and analyzed by CE with laser-induced fluorescencedetection. The electrophoretic separation was carried out in an uncoated capillary with 50μm i.d. and total length of 27 cm (20 cm to the detector) maintained at 25℃under 30kVvoltage. The running buffer was 50mmol/L boric acid buffer and 65g/L PEG 20 000 (pH8.92). the sample buffer was 5mmol/L boric acid buffer(pH 8.74). The linear range of themethod was from 1to 5mg/L. The limit of detection was 0.1 mg/L. The method wassuccessfully applied for the determination of IgG in bovine colostrum products with LIFdetection and achieved satisfactory results.(3) A new CE method was established for the quantitative determination ofα-lactalbumin in milk and milk powders with UV absorption detection at 214nm. Theelectrophoretic separation was carried out in an uncoated capillary with 50μm i.d. andtotal length of 77 cm (70 cm to the detector) maintained at 25℃using 0.5 mol/L boricacid buffer (pH 8.35) under 22 kV voltage. The sample medium was 0.05 or 0.1 mol/Lacetic acid. The linear range of the method was from 50 to 2000 mg/L. The limit ofdetection was 5 mg/L. The method was successfully used to separate a mixture of wheyproteins for the quantitative analysis of theα-lactalbumin in dairy products.