Immobilised Histidine Tagged β (2)-Adrenoceptor Oriented by a Diazonium Salt Reaction and Its Application in Exploring Drug-Protein Interaction Using Ephedrine and Pseudoephedrine as Probes

A new oriented method using a diazonium salt reaction was developed for linking β (2)-adrenoceptor (β (2)-AR) on the surface of macroporous silica gel. Stationary phase containing the immobilised receptor was used to investigate the interaction between β (2)-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 β (2)-AR. At 37 °C, the association constants during the binding were (5.94±0.05)×10(3)/M for ephedrine and (3.80±0.02) ×10(3)/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 β (2)-AR was a spontaneous reaction with exothermal processes. The ΔG(θ), ΔH(θ) and ΔS(θ) for the interaction between ephedrine and β (2)-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 β (2)-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs.