Production of Peroxymonocarbonate by Steady-State Micromolar HO and Activated Macrophages in the Presence of CO/HCO Evidenced by Boronate Probes.

Peroxymonocarbonate (HCO/HOOCO) is produced by the reversible reaction of CO/HCO with HO ( = 0.33 M, pH 7.0). Although produced in low yields at physiological pHs and HO and CO/HCO concentrations, HCO oxidizes most nucleophiles with rate constants 10 to 100 times higher than those of HO. Boronate probes are known examples because HCO reacts with coumarin-7-boronic acid pinacolate ester (CBE) with a rate constant that is approximately 100 times higher than that of HO and the same holds for fluorescein-boronate (Fl-B) as reported here. Therefore, we tested whether boronate probes could provide evidence for HCO formation under biologically relevant conditions. Glucose/glucose oxidase/catalase were adjusted to produce low steady-state HO concentrations (2-18 μM) in Pi buffer at pH 7.4 and 37 °C. Then, CBE (100 μM) was added and fluorescence increase was monitored with time. The results showed that each steady-state HO concentration reacted more rapidly (∼30%) in the presence of CO/HCO (25 mM) than in its absence, and the data permitted the calculation of consistent rate constants. Also, RAW 264.7 macrophages were activated with phorbol 12-myristate 13-acetate (PMA) (1 μg/mL) at pH 7.4 and 37 °C to produce a time-dependent HO concentration (8.0 ± 2.5 μM after 60 min). The media contained 0, 21.6, or 42.2 mM HCO equilibrated with 0, 5, or 10% CO, respectively. In the presence of CBE or Fl-B (30 μM), a time-dependent increase in the fluorescence of the bulk solution was observed, which was higher in the presence of CO/HCO in a concentration-dependent manner. The Fl-B samples were also examined by fluorescence microscopy. Our results demonstrated that mammalian cells produce HCO and boronate probes can evidence and distinguish it from HO under biologically relevant concentrations of HO and CO/HCO.