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Homocysteine-specific fluorescence detection and quantification for evaluating S-adenosylhomocysteine hydrolase activity
Analyst ( IF 4.2 ) Pub Date : 2022-07-07 , DOI: 10.1039/d2an00945e
Chunyu Gao 1 , Ziyi Ding 1 , Jiangkun Tan 1 , Jinmao You 2 , Zan Li 1
Affiliation  

Studies have shown that homocysteine (Hcy) levels are closely related to cardiovascular and cerebrovascular diseases. In this work, we have developed and synthesized three copper complexes, F542-Cu2+, F508-Cu2+, and F465-Cu2+ for Hcy detection. The different binding constants (Ks) of the copper complexes endow them with dramatic reactivity toward biothiols. The pyridine-containing tetraazacycle was employed in the construction of F542-Cu2+, which renders the medium Ks value for the copper complex compared with cyclen and TACN and effectively prevented the disintegration of the complexes. Pyridine-containing tetraazacycle provided the basis and possibility for the hypothesis for the reduction of Cu2+ by biothiols to shape into a stable six-membered ring structure. The obtained results verified that F542-Cu2+ could be utilized to specifically probe Hcy in a switched-on fluorescence mode. F542-Cu2+ exhibited excellent environmental stability, superior sensitivity, and outstanding selectivity toward Hcy under physiological conditions. The mechanism of Hcy specificity was confirmed to be related to the generation of Hcy-induced six-membered ring by fluorescence imaging, time-dependent fluorescence spectra, ESI-MS, and electron paramagnetic resonance (EPR) analyses. Furthermore, we exploited the application of F542-Cu2+ and developed a strategy for evaluating the activity of S-adenosylhomocysteine hydrolase (AHCY) in vitro by fluorescence analysis. More importantly, real-time in vivo evaluation of the enzymatic activity of AHCY was realized and assisted by our probe, providing the possibility of opening up a new avenue for enzymatic reaction assessment.

中文翻译:

用于评估 S-腺苷高半胱氨酸水解酶活性的同型半胱氨酸特异性荧光检测和定量

研究表明,同型半胱氨酸(Hcy)水平与心脑血管疾病密切相关。在这项工作中,我们开发并合成了三种铜配合物F 542 -Cu 2+F 508 -Cu 2+F 465 -Cu 2+用于Hcy检测。铜配合物的不同结合常数 ( Ks ) 赋予它们对生物硫醇的显着反应性。含吡啶的四氮杂环被用于构建F 542 -Cu 2+,这使得介质K s与cyclen和TACN相比,铜配合物的价值并有效地防止了配合物的崩解。含吡啶的四氮杂环为生物硫醇还原Cu 2+形成稳定的六元环结构的假设提供了基础和可能性。所得结果验证了F 542 -Cu 2+可用于在开启荧光模式下特异性探测Hcy。F 542 -Cu 2+在生理条件下表现出优异的环境稳定性、卓越的灵敏度和对 Hcy 的出色选择性。通过荧光成像、时间依赖性荧光光谱、ESI-MS 和电子顺磁共振 (EPR) 分析,证实 Hcy 特异性的机制与 Hcy 诱导的六元环的产生有关。此外,我们利用F 542 -Cu 2+的应用,开发了一种通过荧光分析在体外评估S-腺苷高半胱氨酸水解酶(AHCY)活性的策略。更重要的是,体内实时我们的探针实现并辅助了AHCY酶活性的评估,为酶反应评估开辟了新途径。
更新日期:2022-07-07
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