Kinetic study of NADPH activation using ubiquinone-rhodol fluorescent probe and an Ir^III-complex promoter at the cell...

Nicotine adenine dinucleotide derivatives NADH and NADPH are intimately involved in energy and electron transport within cells. The fluorescent ubiquinone-rhodol (Q-Rh) probe is used for NADPH activation monitoring. Q-Rh reacts with NADPH yielding its quenched hydroquinone-rhodol (H₂Q-Rh) form with concurrent NADPH activation (i.e. NADP⁺ formation). NADPH activation can be enhanced by the addition of an Ir^III-complex (i.e. [(η⁵-C₅Me₅)Ir(phen)(H₂O)]²⁺) as a promoter. The rate of the Q-Rh fluorescence quenching process is proportional to the NADPH activation rate, which can be used to monitor NADPH. Experiments were performed in phosphate-buffered saline (PBS) solution and on HeLa cell cultures to analyze the kinetics of Q-Rh reduction and the influence of the Ir^III-complex promoter on the activation of NADPH (in PBS) and of other intracellular reducing agents (in HeLa cells). There is a substantial increase in Q-Rh reduction rate inside HeLa cells especially after the addition of Ir^III-complex promoter. This increase is partly due to a leakage process (caused by Ir^III-complex-induced downstream processes which result in cell membrane disintegration) but also involves the nonspecific activation of other intracellular reducing agents, including NADH, FADH₂, FMNH₂ or GSH. In the presence only of Q-Rh, the activation rate of intracellular reducing agents is 2 to 8 times faster in HeLa cells than in PBS solution. When both Q-Rh and Ir^III-complex are present, the rate of the Ir^III-complex catalyzed reduction reaction is 7 to 23 times more rapid in HeLa cells. Concentration- and time-dependent fluorescence attenuation of Q-Rh with third-order reaction kinetics (reasonably approximated as pseudo-first-order in Q-Rh) has been observed and modelled. This reaction and its kinetics present an example of "bioparallel chemistry", where the activation of a molecule can trigger a unique chemical process. This approach stands in contrast to the conventional concept of "bioorthogonal chemistry", which refers to chemical reactions that occur without disrupting native biological processes.