Abstract:

Receptor chromatography is an efficient analytical technique that combines the high separation ability of chromatography with the high specificity of receptors for drug recognition. In addition, this technique offers the advantages of active recognition, online separation, and convenient multidimensional target tracking. This strategy allows target active ingredients in complex systems, such as traditional Chinese medicines, to be efficiently screened and accurately identified. Furthermore, the interactions between ligands and immobilized proteins can be studied. To avoid a loss in function, receptor chromatography requires efficient, mild, and simple immobilization methods that do not damage the structure of the immobilized receptors. Improvements in the activity, stability, and ligand-recognition specificity of immobilized functional proteins can be achieved by selecting appropriate immobilization conditions. Notably, the protein immobilization method is not only closely related to the recognition ability of receptor chromatography but also determines the accuracy of the technique.

Common methods for immobilizing functional proteins include physical adsorption, chemical reactions, biological affinity reactions, and click chemistry. Despite being easy to operate under mild reaction conditions, these methods have shortcomings, including poor reaction specificity and the necessity of using high-purity functional proteins to prepare chromatography columns. Maintaining the high activity of immobilized receptors and ensuring excellent identification and separation abilities are key challenges in the further development of receptor chromatography. In this work, these issues were addressed by introducing a specific bioorthogonal reaction involving haloalkane dehalogenase (Halo) and 6-chlorohexanoic acid for the immobilization of the α_1A-adrenergic receptor (α_1A-AR). Specifically, Halo-α_1A-AR was immobilized on the surface of 6-chlorohexanoic acid-modified aminopropyl silica gel in one step.

The stationary phase with immobilized Halo-α_1A-AR was characterized using scanning electron microscopy. Moreover, the activity of the Halo-α_1A-AR chromatographic column was evaluated using specific ligands (terazosin hydrochloride, phentolamine mesylate, tamsulosin hydrochloride, and urapidil) and nonspecific ligands (yohimbe and metoprolol) for α_1A-AR. Halo-α_1A-AR was successfully immobilized on the silica gel surface with good stability over 30 days, and the Halo-α_1A-AR chromatographic column exhibited good ligand-recognition activity. The nonlinear chromatography results indicated that prazosin hydrochloride, terazosin hydrochloride, and urapidil interacted with immobilized Halo-α_1A-AR through one type of binding site, with association constants of 3.85×10⁵, 5.00×10⁵, and 5.90×10⁵L/mol, respectively. In contrast, phentolamine mesylate and tamsulosin hydrochloride interacted with immobilized Halo-α_1A-AR through two types of binding site. The association constants with the high- and low-affinity binding sites were 3.12×10⁶ and 6.01×10⁵L/mol, respectively, for phentolamine mesylate and 9.98×10⁵ and 0.21×10⁵L/mol, respectively, for tamsulosin hydrochloride.

Compared with the traditional carbonyldiimidazole method, the immobilization method developed in this work did not require receptor purification and thus minimized the loss of receptor activity. The affinity constants obtained with immobilized Halo-α_1A-AR were consistent with the values determined for receptor-ligand binding in solution, indicating that the Halo-α_1A-AR chromatography column is suitable for studying drug-protein interactions. This approach also provides a foundation for the efficient screening and accurate determination of target active ingredients in complex systems.

Key words: α_1A-adrenergic receptor (α_1A-AR), haloalkane dehalogenase (Halo), one-step immobilization, nonlinear chromatography