Homocysteine-specific fluorescence detection and quantification for evaluating S-adenosylhomocysteine hydrolase activity

Abstract

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 (K_s) 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 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 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.