卤代烷烃脱卤素酶标签α1A-肾上腺素受体色谱方法的建立与评价

Establishment and evaluation of haloalkane dehalogenase taggedα1A-adrenergic receptor chromatography

受体色谱技术是将色谱技术的高分离能力和药物-受体间的高特异性识别能力结合起来的一种分析技术,能够对中药等复杂体系中靶向活性成分进行高效筛选与准确辨识。该技术的关键在于发展高效、温和、简便的固定化方法,使得固定化受体活性得以保持。传统的以生物交联剂为核心的随意共价固定化技术存在反应特异性较差、需要纯化蛋白质等不足。针对该问题,本研究将卤代烷烃脱卤素酶(Halo)与6-氯己酸的特异性生物正交反应引入至α1A-肾上腺素受体(α1A-AR)的固定化过程,将Halo标签α1A-AR一步固定至6-氯己酸修饰的氨丙基硅胶表面,无需对α1A-AR进行纯化。采用扫描电子显微镜和色谱法对Halo-α1A-AR色谱固定相进行形貌及活性表征,证明受体已成功固定且具有特异性识别配体的能力,30天内稳定性良好。非线性色谱法研究结果显示:盐酸哌唑嗪、盐酸特拉唑嗪和乌拉地尔通过一类结合位点与Halo-α1A-AR色谱固定相作用,结合常数分别为3.85×10^(5)、5.00×10^(5)和5.90×10^(5)L/mol;甲磺酸酚妥拉明和盐酸坦索罗辛在Halo-α1A-AR色谱固定相上则存在两类位点,前者与受体亲和力分别为3.12×106 L/mol和6.01×10^(5)L/mol,后者则为9.98×10^(5)L/mol和2.11×104 L/mol。与传统物理吸附法或N,N′-羰基二咪唑法制备的α1A-AR色谱柱相比,本文所用固定化方法无需纯化受体,在一定程度上避免了受体活性损失,实现了蛋白质一步固定化方法,亲和力测定值更接近于溶液中受体-药物的真值,为复杂体系靶向活性成分高效筛选与准确测定奠定了基础。

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 theα1A-adrenergic receptor(α1A-AR).Specifically,Halo-α1A-AR was immobilized on the surface of 6-chlorohexanoic acid-modified aminopropyl silica gel in one step.The stationary phase with immobilized Halo-α1A-AR was characterized using scanning electron microscopy.Moreover,the activity of the Halo-α1A-AR chromatographic column was evaluated using specific ligands(terazosin hydrochloride,phentolamine mesylate,tamsulosin hydrochloride,and urapidil)and nonspecific ligands(yohimbe and metoprolol)forα1A-AR.Halo-α1A-AR was successfully immobilized on the silica gel surface with good stability over 30 days,and the Halo-α1A-AR chromatographic column exhibited good ligand-recognition activity.The nonlinear chromatography results indicated that prazosin hydrochloride,terazosin hydrochloride,and urapidil interacted with immobilized Halo-α1A-AR through one type of binding site,with association constants of 3.85×10^(5),5.00×10^(5),and 5.90×10^(5)L/mol,respectively.In contrast,phentolamine mesylate and tamsulosin hydrochloride interacted with immobilized Halo-α1A-AR through two types of binding site.The association constants with the high-and low-affinity binding sites were 3.12×106 and 6.01×10^(5)L/mol,respectively,for phentolamine mesylate and 9.98×10^(5)and 0.21×10^(5)L/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-α1A-AR were consistent with the values determined for receptor-ligand binding in solution,indicating that the Halo-α1A-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.