title: Immobilised histidine tagged β2-adrenoceptor oriented by a diazonium salt reaction and its application in exploring drug-protein interaction using ephedrine and pseudoephedrine as probes.
abstract: A new oriented method using a diazonium salt reaction was developed for linking β2-adrenoceptor (β2-AR) on the surface of macroporous silica gel. Stationary phase containing the immobilised receptor was used to investigate the interaction between β2-AR and ephedrine plus pseudoephedrine by zonal elution. The isotherms of the two drugs best fit the Langmuir model. Only one type of binding site was found for ephedrine and pseudoephedrine targeting β2-AR. At 37 °C, the association constants during the binding were (5.94±0.05)×103/M for ephedrine and (3.80±0.02) ×103/M for pseudoephedrine, with the binding sites of (8.92±0.06) ×10-4 M. Thermodynamic studies showed that the binding of the two compounds to β2-AR was a spontaneous reaction with exothermal processes. The ΔGθ, ΔHθ and ΔSθ for the interaction between ephedrine and β2-AR were -(22.33±0.04) kJ/mol, -(6.51±0.69) kJ/mol and 50.94±0.31 J/mol·K, respectively. For the binding of pseudoephedrine to the receptor, these values were -(21.17±0.02) kJ/mol, -(7.48±0.56) kJ/mol and 44.13±0.01 J/mol·K. Electrostatic interaction proved to be the driving force during the binding of the two drugs to β2-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs.
authors.affiliation: College of Life Sciences, Northwest University, Xi'an, Shaanxi, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Li
authors.affiliation: College of Life Sciences, Northwest University, Xi'an, Shaanxi, China.
authors.forename: Liujiao
authors.initials: L
authors.lastname: Bian
authors.affiliation: College of Life Sciences, Northwest University, Xi'an, Shaanxi, China.
authors.forename: Xinfeng
authors.initials: X
authors.lastname: Zhao
authors.affiliation: College of Life Sciences, Northwest University, Xi'an, Shaanxi, China.
authors.forename: Xiaokang
authors.initials: X
authors.lastname: Gao
authors.affiliation: Institute of Analytical Science, Northwest University, Xi'an, Shaanxi, China.
authors.forename: Jianbin
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Institute of Vascular Medicine, Peking University; Third Hospital and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing, China.
authors.forename: Zijian
authors.initials: Z
authors.lastname: Li
authors.affiliation: Institute of Vascular Medicine, Peking University; Third Hospital and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing, China.
authors.forename: Youyi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: School of Pharmacy, The Fourth Military Medical University, Xi'an, Shaanxi, China.
authors.forename: Ru
authors.initials: R
authors.lastname: Jiang
authors.affiliation: College of Life Sciences, Northwest University, Xi'an, Shaanxi, China.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Zheng
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D003979:Diazonium Compounds / Q000737:chemistry*; D004809:Ephedrine / Q000378:metabolism*; D006639:Histidine; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D062105:Molecular Docking Simulation; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D054199:Pseudoephedrine / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000378:metabolism*; D012492:Salts / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D003979:Diazonium Compounds; D055767:Immobilized Proteins; D018343:Receptors, Adrenergic, beta-2; D012492:Salts; D006639:Histidine; D012822:Silicon Dioxide; D054199:Pseudoephedrine; D004809:Ephedrine
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