| The interaction of small molecules with proteins is important in many biological processes. Protein binding in blood is significant in determining the eventual activity and fate of drugs once they have entered the circulation. These interactions help control the distribution, rate of excretion, and toxicity of drugs in the body. They include the binding interaction between one drug and protein as well as direct or indirect competition between two drugs for the same binding protein. High-performance affinity chromatography is a powerful technique, which has received a great progress in studying the interactions between drugs and proteins. However, there are still many problems in this method to be solved. For instance, fewer methods in preparation of high activity protein stationary phase, insufficient study on the interaction between drugs and target proteins and less study on the drug-drug competition with one protein. To solve these problems, a method of oriented immobilization of β2-adrenergic receptor (β2-AR, one of the receptor proteins) was developed and applied in the study of the interaction between drugs and β2-AR. In addition, human serum albumin (HSA) was chosen as a model protein to investigate the competitive interactions between the bioactive components of Chinese herbal medicine and protein. This dissertation is divided into five chapters:1. IntroductionThe significance, content and methods for studying interaction between drugs and proteins were introduced. In addition, the principle of high-performance affinity chromatography (HPAC), methods of preparation high-performance affinity chromatography stationary phase, theories of high-performance affinity chromatography and their application in the interactions of drug-protein were reviewed.2. Preparation and characterization of oriented immobilization of β2-adrenergic receptorIn order to prepare highly active β2-AR stationary phase, a novel oriented immobilized method of β2-AR was developed according to the character of β2-AR molecular structure. In terms of the total β2-AR content, specific binding and binding capacity, it was proved that β2-AR column prepared by the oriented method had a better results than the β2-AR column immobilized to the silica through a random immobilization method. It provided a novel process to the preparation of highly active protein stationary phase.3. The interaction of drugs with oriented immobilized β2-adrenergic receptorThe interactions of propranolol, salbutamol sulphate and clorprenaline with oriented immobilized β2-AR were investigated respectively by the frontal analysis and the zonal elution. The binding isotherms of these three drugs on the β2-AR column were determined by frontal analysis at pH7.4and37C. It was showed that the three drugs had only one kind of binding site on the β2-AR molecular, and the association constants of propranolol, salbutamol sulphate and clorprenaline were1.96×104L/mol,5.78×103L/mol and6.07x103L/mol, respectively. Meanwhile, the competition interactions of these three drugs with β2-AR were determined by zonal elution. It was indicated that these three drugs competed the same binding site on β2-AR molecular and the association constants of salbutamol sulphate and clorprenaline with β2-AR were9.17×103L/mol and2.94×103L/mol, respectively. The association constants of three drug determined by zonal elution were similar with the results of frontal analysis. This research demonstrated that oriented immobilized β2-AR can be applied well in the interaction of drugs with β2-AR.4. The competitive interaction of three bioactive components in Danshen injection with human serum albuminThe interactions of three bioactive components (danshensu, caffeic acid and protocatechuic aldehyde) in Danshen injection with HSA had been studied by the HPAC, microdialysis-HPLC and molecular docking method.When the single component of Danshen injection with HSA was studied, the binding isotherms of protocatechuic aldehyde and caffeic acid were determined by frontal analysis. The results showed that both of the two components had only one kind of binding site on HSA. The association constants of protocatechuic aldehyde and caffeic acid with HSA were determined at pH7.4,37C by self-competition zonal elution, and the association constants were9.56×103L/mol and1.60×104L/mol, respectively. The binding interaction of single component with HSA was also investigated by microdialysis-HPLC. It was showed that the single component also had one kind of binding site on HSA in solution, corresponded with the results determined by HPAC. The association constants for danshensu, protocatechuic aldehyde and caffeic acid with HSA determined by microdialysis-HPLC were1.27×105L/mol,4.03×104L/mol and2.75×104L/mol, respectively.When the multicomponents of Danshen injection with HSA were studied, the results of zonal elution showed that the three components had a direct competition on HSA. The association constants for danshensu, protocatechuic aldehyde and caffeic acid determined by zonal elution were3.56×103L/mol,2.44×104L/mol and1.85×104L/mol, respectively. Microdialysis-HPLC was applied to study the competition of three components with HSA, and the results were consistent with zonal elution studies.Further study was performed to investigate the binding site of the three components on HSA molecular. Zonal elution studies with the probes of HSA as injected solutes showed that three components were binding to indole-benzodiazepine site on HSA molecular. Thermodynamic results indicated that the interactions between protocatechuic aldehyde and caffeic acid with HSA were mainly drived respectively by hydrophobic interaction and electrostatic force.5. A new method to determine association constants of protein-drug interactionAn equation describing the relationship between the injected amount of solutes and their retention values in liquid chromatography was introduced into drug-protein study. A new method to determine the association constant of drug-protein interaction was developed. This method was applied in studying the interaction of propranolol, salbutamol sulphate and clorprenaline with β2-AR. The association constants for propranolol, salbutamol sulphate and clorprenaline were2.01×104L/mol,2.52×103L/mol and8.19×103L/mol, respectively. Compared with the results of HPAC, it was indicated that this method is a simple and reliable method in drug-protein study. |
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