| Serum Albumin is the most abundant carrier protein in blood circulation. The investigation on the binding mechanism of drugs with serum albumin is of importance in toxicology and pharmacokinetics. In this dissertation, the affinity chromatography combined with fluorescence spectroscopy was used to investigate the interaction of three drugs, berberine chloride, chlorpromazine and propranolol, with bovine serum albumin (BSA). The two major works were carried out as in the following:Firstly, frontal analysis was used to examine the interaction of berberine chloride, chlorpromazine and propranolol with immobilized BSA. The influence of temperature, pH and 1-propanol content in the mobile phase on the capacity factor (k'), association constant (K) and moles of binding sites (m_L) for the three drugs on an immobilized BSA column had also been investigated. The conclusions could be got:(1) At 30℃, the binding constants of berberine chloride, chlorpromazine and propranolol with immobilized BSA were 4.79×10~4, 2.67×10~4 and 4.21×10~4L/mol respectively, and all the percentage protein binding of the three drugs were more than 80%, indicating that the interaction forces between the three drugs and BSA were strong, and all of the three drugs could be reserved and transmitted by BSA.(2) As the temperature was heightened, the retention of all the three drugs on BSA column decreased. This phenomena was mainly caused by the depression of both association constant (K) and moles of binding sites (m_L), while the changes in m_L accounted for most of the shifts in retention of chlorpromazine.(3) During the binding process of berberine chloride and propranolol with BSA, the thermodynamic parameters,△G＜0,△S＞0 and△H＜0, suggested that the main driving force of their interaction with BSA was electrostatic force. While for the association between chlorpromazine and BSA,△G＜0,△H＞0,△H＞0, indicating that hydrophobic force was the main driving force.(4) As the pH of the mobile phase was heightened, the binding constants of chlorpromazine and propranolol with BSA increased. When content of 1-propanol in the mobile phase increased, the binding constants of the two drugs decreased, which indicated the hydrophobic force had some influences on the binding of the two drugs with BSA. Moreover, the magnitude in the decrease of the association constants of chlorpromazine was more than that of propranolol, which may be consistent with the conclusion that hydrophobic force was the main driving force for the binding of chlorpromazine with BSA.Secondly, the interaction of berberine chloride and chlorpromazine with BSA was further investigated by fluorescence spectroscopy, and the result was also compared to that obtained by the frontal analysis. The conclusions could be drawn as following:(1) The fluorescence quenching of berberine chloride and chlorpromazine with BSA was thought to be deduced by combining static quenching with nonradiative energy transfer. At 40℃, the apparent association constants of berberine chloride and chlorpromazine with BSA were 6.56×10~4 and 1.45×10~4L/mol, respectively, which are close to the corresponding ones obtained by the frontal analysis.(2) The stereo-distance (r) of berberine chloride and chlorpromazine with fluorescent amino acid residues of the BSA was 1.89 and 1.27nm at 20℃, respectively, which affirmed that part of the two drugs segment had inserted into the hydrophobic pocket of BSA. The thermodynamic analysis suggested that the main driving force for the interaction between berberine chloride and BSA was electrostatic force, while for the interaction between chlorpromazine and BSA was hydrophobic force.(3) Via comparing the binding constants and thermodynamic parameters attained by frontal analysis and fluorescence spectroscopy, it could be concluded that the binding behavior of the two drugs with immobilized BSA was similar to that with BSA in solution. |
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