Quantitative assessment of the in-vitro binding kinetics of antisickling aromatic aldehydes with hemoglobin A: A universal HPLC-UV/Vis method to quantitate Schiff-base adduct formation.
Quantitative assessment of the in-vitro binding kinetics of antisickling aromatic aldehydes with hemoglobin A: A universal HPLC-UV/Vis method to quantitate Schiff-base adduct formation.
复制标题
抗镰化芳香醛与血红蛋白 A 的体外结合动力学的定量评估：定量席夫碱加合物形成的通用 HPLC-UV/Vis 方法。
DOI：
10.1016/j.jpba.2022.115152
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发表时间：
2023
影响因子：
3.4
通讯作者：
Venitz,Jürgen
中科院分区：
文献类型：
--
作者：
Xu,Xiaomeng;Ghatge,MohiniS;Huang,Boshi;Alghamdi,Ahmed;Wang,Huiqun;Pierce,BDaniel;Abdulmalik,Osheiza;Zhang,Yan;Safo,MartinK;Venitz,Jürgen
芳香醛作为血红蛋白（AEH）的变构效应物，与蛋白质形成席夫碱加合物，以增加其氧（O2）亲和力;这是镰状细胞病（SCD）治疗中的理想特性，因为高O2亲和力血红蛋白（Hb）不会使红细胞镰状化，从而防止红细胞镰状化。本研究报告了弱阳离子交换HPLC测定法的开发、验证和应用-定量Hb-AEH加合物的出现-作为一种“通用”方法，允许通过了解其Hb结合亲和力和动力学来优先考虑AEH候选物。用HPLC测定了10种AEH的浓度和时间依赖性Hb结合曲线，然后进行适当的非线性建模以表征其稳态结合亲和力（KDss）和结合动力学二级缔合（kon）和一级解离（koff）速率常数。香草醛衍生的AEH表现出增强的结合亲和力的血红蛋白，主要是由于他们更快的kon。在AEH中，kon和koff值强烈相关（r = 0.993，n = 7），表明AEH支架的修饰如预期的那样增强了它们与Hb的相互作用，但无意中增加了它们的Hb-AEH加合物解离。据我们所知，本研究是第一个提供有价值的洞察血红蛋白结合动力学的抗镰状芳香醛，和测定将是一个有用的平台，在筛选/优先考虑候选药物SCD治疗。
Aromatic aldehydes act as allosteric effectors of hemoglobin (AEH), forming Schiff-base adducts with the protein to increase its oxygen (O2) affinity; a desirable property in sickle cell disease (SCD) treatment, as the high-O2affinity hemoglobin (Hb) does not polymerize and subsequently prevents erythrocytes sickling. This study reports the development, validation, and application of a weak cation-exchange HPLC assay – quantifying the appearance of Hb-AEH adduct – as a “universal” method, allowing for the prioritization of AEH candidates through an understanding of their Hb binding affinity and kinetics. Concentration- and time-dependent Hb binding profiles of ten AEHs were determined with HPLC, followed by the appropriate non-linear modeling to characterize their steady-state binding affinity (KDss), and binding kinetics second-order association (kon) and first-order dissociation (koff) rate constants. Vanillin-derived AEHs exhibited enhanced binding affinity to Hb, primarily due to their faster kon. Across AEH, konand koffvalues are strongly correlated (r = 0.993, n = 7), suggesting that modifications of the AEH scaffold enhanced their interactions with Hb as intended, but inadvertently increased their Hb-AEH adduct dissociation. To our knowledge, the present study is the first to provide valuable insight into Hb binding kinetics of antisickling aromatic aldehydes, and the assay will be a useful platform in screening/prioritizing drug candidates for SCD treatment.