乙酰唑胺与牛血清白蛋白相互作用的表面等离子体共振结合研究

IF 0.3 Q4 MEDICINE, RESEARCH & EXPERIMENTAL Clinical and Experimental Health Sciences Pub Date : 2023-04-11 DOI:10.33808/clinexphealthsci.1218956
Emir Alper Türkoğlu, Fatma Gülruy Aydin
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引用次数: 0

摘要

目的:血清白蛋白是体循环中主要的血浆蛋白,是内源性和外源性化合物(如药物)的转运蛋白。在制药应用中,在评估候选药物时,表征药物如何与血清白蛋白结合是至关重要的。表面等离子体共振(SPR)是一种快速、实时、无标记的基于光学的检测技术,可以实时、高灵敏度地监测分子相互作用、分析结合反应和确定亲和常数。乙酰唑胺(AZA)用于治疗癫痫和青光眼。方法:为确定AZA-牛血清白蛋白(BSA)相互作用的结合动力学,(i)将SPR金传感器表面功能化,(ii)采用胺偶联程序激活表面基团,将BSA固定在功能化的传感器表面,(iii)将AZA的浓度系列(10、25、50、75、100、150、200和250µM)注射到SPR系统中,(iv)利用SPR系统软件测量动力学值。结果:5 mM MUA包被表面功能化。以250µg/mL BSA为配体,流速为30µL/min, 1X PBS缓冲液(pH 7.4)和10 mM乙酸缓冲液(pH 5.2)分别作为运行缓冲液和偶联缓冲液进行SPR结合研究。结果表明,AZA-BSA的平衡常数(KD)为67.72µM。结论:本研究利用基于Kretchmann构型的SPR体系研究了AZA-BSA的结合相互作用。该研究采用快速、无标签和实时的方法设计,将为制药和临床应用提供有价值的知识。
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Surface Plasmon Resonance Binding Study on the Interaction of Acetazolamide and Bovine Serum Albumin
Objective: Serum albumins are major plasma proteins in systemic blood circulation and act as transport proteins for endogenous and exogenous compounds such as drugs. In pharmaceutical applications, it is essential to characterize how drugs bind to serum albumin in the evaluation of drug candidates. Surface plasmon resonance (SPR) is fast, real-time, label-free optical based detection technique that offers the monitoring of molecular interactions, analyzing binding reactions and determining the affinity constants with real-time and high sensitivity. Acetazolamide (AZA) is used in the treatment of epilepsy and glaucoma. Methods: To determine the binding kinetics of AZA-Bovine serum albumin (BSA) interaction, (i) SPR gold sensor surface was functionalized, (ii) amine coupling procedure was applied to activate the surface group and BSA was immobilized on functionalized sensor surface, (iii) the concentration series of AZA (10, 25, 50, 75, 100, 150, 200 and 250 µM) was injected to SPR system and (iv) kinetic values were measured using the software of SPR system. Results: 5 mM MUA was coated for surface functionalization. 250 µg/mL BSA as ligand, 30 µL/min flow rate, 1X PBS buffer (pH 7.4) and 10 mM acetate buffer (pH 5.2) as running and coupling buffers, respectively, were performed for SPR binding study. According to result, equilibrium constant (KD) of AZA-BSA was determined as 67.72 µM. Conclusion: In this study, we investigated the AZA-BSA binding interaction using SPR system based on Kretchmann configuration. The study designed with fast, label-free and real-time approach will provide valuable knowledge for pharmaceutical and clinical applications.
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Clinical and Experimental Health Sciences
Clinical and Experimental Health Sciences MEDICINE, RESEARCH & EXPERIMENTAL-
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