| The interaction between functional proteins and small molecular ligands has very important significance for revealing the complex signal transmitting involved in the process of life activities and the development of innovative drugs. Biological sensor, reagent box, enzyme reactor and affinity chromatography method are the main ways to achieve the above research. However, due to the fixed interface protein orientation and conformation disorder, the results from the physiological state of the functional protein and drug interaction studies are different. Therefore, the research on the orientation and conformation regulation methods of the functional proteins is one of the frontiers in the field of medicine, chemistry and biology. In the previous work, the authors established the method of beta2-adrenergic receptor chromatography. The method can be used to study the interaction between receptors and drugs, and to screen the active constituents. Based on this, this thesis is aimed at the problem of the surface receptor orientation and conformation disorder of the stationary phase, and study on the establishment and application of the method for the establishment and application of the immobilized beta2-adrenergic receptor orientation and conformation. This study is expected to provide the basis for the establishment of a high sensitive method for other functional proteins, which is of great significance for the development of high efficiency and innovative drugs. The full text is divided into four chapters and the author’s main contribution is as follows:(1)A new method for the directed immobilization of beta2-adrenergic receptors was established, and the model of regulating orientation and conformation of beta2-adrenergic receptor was constructed. The histidine tag in the structure of beta2-adrenergic receptor was used to specific binding of metal nickel ion to the stationary phase, which was also directed at the macroporous silica surface. The results showed that the proposed method can obtain a fixed receptor for orientation. The medicine clorprenaline hydrochloride as the ligand, and specificity and stability of the receptor chromatography model were characterized. On this basis, ephedrine hydrochloride and pseudoephedrine hydrochloride as conformation dependent probe, by changing the temperature, mobile phase composition and ligand conditions, the conformation of the receptor is regulated. The results show that the most stable conformation of the immobilized receptor can be obtained when the temperature is 25.0℃; When the concentration of phosphate buffer solution of mobile phase was 5.0mmol/L (pH=7.40), the conformation of beta2-adrenergic receptor was the best; take (S)-propranolol as ligand, the effect of drug ligands on the conformation of the fixed beta2-adrenergic receptor was investigated, this ligand can induce the change of the conformation of the receptor and the concentration of 5μmol/L propranolol can make the immobilized beta2-adrenergic receptor to obtain optimal conformation. This study provided a methodological reference for other functional proteins to regulate the orientation and conformation.(2) The interaction of seven kinds of drugs, such as the receptor and the terbutaline sulfate, was studied by using the stable state of the model of the receptor. The binding constants and binding sites of the interaction between terbutaline sulfate, methoxyphenamine hydrochloride, clorprenaline hydrochloride, tulobmerol hydrochloride, salbutamol sulfate, clenbuterol hydrochloride, bambuterol hydrochloride and the beta2-adrenaline receptor were determined by frontal chromatography. The results showed that there was a class of binding sites for the seven drugs and beta2-adrenaline receptors and the binding constant was: 1.84×104 M-1, 1.71×104 M-1,2.3×104 M-1,2.×104 M-1,1.98×104 M-1,2.37×104 M-1 and 9.43×104 M-1; the number of binding sites was:8.3×10-4 M,3.88×10-5 M,1.02×10-5 M, 2.16×10-5 M,7.5×10-5 M,1.82×10-5 M,2.47×10-5 M. By using microdialysis to verify the results, the results showed that two methods is the same amount as the binding constant and the number of binding sites. It is proved that the beta2-adrenergic receptor chromatography model can be used to study the interaction between drug molecules and receptors, which provides a reference for the high throughput analysis of online protein and drug interactions.(3) Application of stable conformation of beat2-adrenergic receptor chromatography models were screened for corydalis and stemona active ingredients, the mechanism of active components and receptors was studied by the method of frontal chromatography and zonal chromatography. Heating reflux preparation of corydalis and stemona of the total extract, receptor chromatography analysis, the structure identification of the collection and retention components by reversed-phase liquid chromatography and mass spectrometry was carried out. It was found that tetrahydro berberine, opioid alkaloid, corydaline and four-hydrogen-columbamine-corydalis were the active ingredients of corydalis which have specific effect with beta2-adrenergic receptor; stemoninine was the active ingredient of stemona which has specific effect with beta2-adrenergic receptor. Further by frontal chromatography measured the binding constants of tetrahydroberberine and protopine were respectively 9.04×104M-4 and 4.30×104 M-1, the binding sites were 6.67× 10-4 M and 5.88×10-4 M, The effect of these two components on β2-AR was studied by the method of zonal elution, It is proved that the binding sites of the two components with beta2-adrenergic receptor were APS.113 of the third hydrophobic regions, which provides a method for the selection of active components in complex samples, which is of great significance for the development of high efficiency and innovative drugs. |
PDF Full Text Request