Site-selective covalently immobilized alpha 1A adrenergic receptor for thermodynamic and extra-thermodynamic study of four ligands binding to the receptor by chromatographic methods.

Immobilized G protein-coupled receptor is a versatile tool to study ligand-receptor interactions. In this work, we synthesized the immobilized alpha 1A adrenergic receptor (α-AR), a GPCR subtype mediating smooth muscle contraction, through a site-selective covalent method that relies on the reaction between haloalkane dehalogenase tagged α-AR and macroporous silica gel coated with 6-chlorohexanoic acid. To investigate thermodynamic and extra-thermodynamic parameters for ligand binding, we utilized the covalently immobilized receptor as stationary phase to perform frontal analysis and injection-amount dependent analysis as well as compared with the random immobilization method. Terazosin gave the association constant of 1.48 × 10 M to α-AR, indicating that the oriented immobilization of α-AR enhances the ligand-binding activity by one order of magnitude in comparison with the random immobilization method (7.9 × 10 M). The binding of phentolamine and tamsulosin to the receptor was accompanied by a large absolute heat capacity (ΔC) of 1.28 ± 0.23 kJ mol, demonstrating that the binding enthalpy and entropy appear to compensate for one another. These results indicated that the covalent immobilization of the receptor onto solid support has a profound impact on the ligand-binding activity of the receptor and the determination of ligand-receptor binding parameters. The receptor immobilized through the site-selective method will act as a benchmark for chromatographic determination of binding parameters in ligand-receptor interactions and can be used as an effective approach for rapid analysis of drug-protein interactions with high accuracy.