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

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 (H 2 Q-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. [(η 5 -C 5 Me 5 )Ir(phen)(H 2 O)] 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, FADH 2 , FMNH 2 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.