Investigation On The Immobilized β₂-adrenceptor And Drugs By Affinity Chromatography

Receptors are the main targets of drugs in vivo.Studying on the drug-receptor interaction has important significance in understanding interaction between the molecular mutual recognition process in life science,medicine and pharmacy.β₂-adrenceptor is one of the members of a family of seven transmembrane proteins coupled to G-proteins,and it is the main target of drugs for cough and asthma.In this thesis,the interaction between six drugs andβ₂-AR was investigated with immobilizedβ₂-adrenceptor affinity chromatography,and the main results are in the following:1.β₂-AR was purified from the rabbit lung tissue by sepharose-salbutamol affinity chromatographic column.To prepare theβ₂-AR stationary phase,β₂-AR was evenly immobilized on the surface of macro-pore silica with a mild chemical coupling method through covalent bond.Binding constants of ligand-receptor with fours ligands adrenaline hydrochloride,noradrenaline bitartrate,propranolol hydrochloridea and salbutamol sulfate were determined by frontal chromatography.The result showed that the order of the association constants for the four ligands was accorded with that obtained by radioimmunoassay,indicating that immobilizedβ₂-AR after purifying,chemical bonding and packing procedure been still its original bioactivity.2.The binding characteristics between palmatine,jatrorrhizine andβ₂-AR were investigated with zonal elution.These results indicated that palmatine had one common binding site on the immobilizedβ₂-AR column,the association constant was 2.93×10⁴ L/mol at pH 7.2 and 25℃,while jatrorrhizine had two bingding sites,the association constants at these two sites were 1.93×10⁴ and 1.56×10⁵ L/mol,respectively.And the molar ratio of weak bingding sites to strong binding sites was 93:7.Palmatine and jatrorrhizine had competed for low-affinity site in immobilizedβ₂-AR.3.Effects of temperature and mobile phase composition on the interaction betweenβ₂-AR and both of palmatine and jatrorrhizine.The results indicated that the interaction between the two drugs andβ₂-AR was mainly drived by electrostatic action,while hydrophobic interaction played minor role in the interaction.4.The total extracts of Semen Armeniacae Amarum were screened by immobilizedβ₂-AR affinity chromatography,and the obtained active compound was to be Amygdalin by LC-MS.The results indicated that immobilized receptor chromatography was a new way to targetly screen the active compound from Traditional Chinese Medicine...