Studies Of The Interaction Of Micromolecule With Serum Albumin Using Various Methods

The research actuality of interaction between micromolecules with serum albumin in recent years has been reviewed and the interaction between some small molecule serum albumin (SA) was studied. Several methods have become firmly entrenched, particularly for the interaction of small molecules and macromolecules. However, the spectroscopy investigation combined with chemometrics in this field is limited. The application of chemometrics in complicated biochemical system can solve some problems and obtain the equilibrium concentration and pure spectra of each component.This thesis is divided into five parts:PartⅠIn part one, firstly, the structures, functions and natures of SA were introduced. Then the methods and contents of interaction of different kind of small ligands with serum albumin were reviewed in turns. The author listed some experimental results of small molecile with serum albumin. Finally, the existing problems and trends of research were discussed.Part ⅡIn part two, the interaction between dobutrex and bovine serum albumin (BSA) was studied by fluorescence spectra under the simulated physiological condition. It was shown that this compound has a quite strong ability to quench the intrinsic fluorescence of BSA. The quenching mechanism was static quenching procedure. The fluorescence quenching data were analyzed according to Stern-Volmer equation and Line weaver-Burk equation, thermodynamic parameters, binding constants Ka, the number of binding sites n were evaluated at298,301and304K, respectively. The main sorts of acting force between the drug and BSA was found to be hydrophobic force. The site markers competitive experiments indicated that the binding of dobutrex and bovine serum albumin primarily in site Ⅱ. The effect of dobutrex on the conformation of BSA was analyzed by synchronous fluorescence.PartⅢIn part three, the interaction of aspirin and ibuprofen with bovine serum albumin (BSA) was studied by spectrofluorimetry under simulated physiological conditions. Both aspirin and ibuprofen quenched the intrinsic fluorescence of BSA and the binding ratios obtained were2:1for aspirin-BSA and3:1for ibuprofen-BSA interactions, respectively. The thermodynamic parameters (AH, AS and AG) obtained from the fluorescence spectroscopy data showed that the binding of aspirin to BSA involved electrostatic interactions. Competitive experiments using warfarin and diazepam as site marker, indicated that aspirin was mainly located in the hydrophobic pocket of site II of the protein as well as to a small extent in site I. Furthermore, the competitive interaction of the aspirin and ibuprofen with BSA, which was studied with the use of the three-way excitation-emission fluorescence spectra and parallel factor analysis (PARAFAC) chemometrics method, showed that the competitive effect of ibuprofen was stronger than that of aspirin i.e. the former molecule replaced the aspirin from the aspirin-BSA complex. PartⅣIn part four, interaction between carcinogens, bendamustine (BDM) and dexamethasone (DXM), with BSA was investigated under pseudo-physiological conditions (Tris-HCl buffer of pH7.4) and with the use of fluorescence and UV-vis spectroscopies. The static mechanism was found to be responsible for the quenching of the fluorescence during such interactions; the binding constant accompanying the formation of the BSA-BDM complex and the binding number were5.14×105L mol-1and1.0, respectively. Spectroscopic studies of the progress of the reaction for the formation of BDM-BSA complex were interpreted with the use of the multivariate curve resolution-alternating least squares (MCR-ALS), which supported the formation of this complex. The site marker treated BSA samples (warfarin (site I) and ibuprofen (site II)) were reacted separately with BDM and DXM; while both anti-carcinogens bound to site I, the estimates of the binding constants suggested that DXM formed a somewhat more stable complex. The concentration profiles of the fluorescence associated with BDM, DXM and BSA, were recovered simultaneously with the use of the PARAFAC chemometrics method, it was demonstrated that as the DMX compound was added to the BDM-BSA complex, free BDM was released being replaced in the complex by DXM.PartⅤIn part five, four antioxidants interaction with BSA was investigated under pH=7.4, we were used several kinds of methods, such as UV-vis spectrophotometry, fluorescence spectroscopy, FT-IR and electrochemical. During a series of experiments can get that the different results, Alough four antioxidants which have similar structure and function. With the use of UV-vis spectrophotometry was found the interaction between BSA and four antioxidants, respectively. Application fluorescence got more evidence to determine the interaction between them. FT-IR got four antioxidant supplements secondary structure change on serum albumin, the conclusion was BHA>BHT>PG>TBHQ. At last, UV-vis and fluorescence spectroscopy study of the progress of the reaction for the formation of antioxidant-BSA complex were interpreted by MCR-ALS, which supported the formation of this complex and the different during four antioxidants.