Homocysteine-specific fluorescence detection and quantification for evaluating S-adenosylhomocysteine hydrolase activity
ANALYST(2022)
摘要
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, F-542-Cu2+, F-508-Cu2+, and F-465-Cu2+ for Hcy detection. The different binding constants (K-s) of the copper complexes endow them with dramatic reactivity toward biothiols. The pyridine-containing tetraazacycle was employed in the construction of F-542-Cu2+, which renders the medium K-s 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 F-542-Cu2+ could be utilized to specifically probe Hcy in a switched-on fluorescence mode. F-542-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 F-542-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.
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