Production of Peroxymonocarbonate by Steady-State Micromolar H2O2 and Activated Macrophages in the Presence of CO2/HCO3– Evidenced by Boronate Probes

Abstract

Peroxymonocarbonate (HCO₄^–/HOOCO₂^–) is produced by the reversible reaction of CO₂/HCO₃^– with H₂O₂ (K = 0.33 M^–1, pH 7.0). Although produced in low yields at physiological pHs and H₂O₂ and CO₂/HCO₃^– concentrations, HCO₄^– oxidizes most nucleophiles with rate constants 10 to 100 times higher than those of H₂O₂. Boronate probes are known examples because HCO₄^– reacts with coumarin-7-boronic acid pinacolate ester (CBE) with a rate constant that is approximately 100 times higher than that of H₂O₂ and the same holds for fluorescein-boronate (Fl-B) as reported here. Therefore, we tested whether boronate probes could provide evidence for HCO₄^– formation under biologically relevant conditions. Glucose/glucose oxidase/catalase were adjusted to produce low steady-state H₂O₂ 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 H₂O₂ concentration reacted more rapidly (∼30%) in the presence of CO₂/HCO₃^– (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 H₂O₂ concentration (8.0 ± 2.5 μM after 60 min). The media contained 0, 21.6, or 42.2 mM HCO₃^– equilibrated with 0, 5, or 10% CO₂, 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 CO₂/HCO₃^– in a concentration-dependent manner. The Fl-B samples were also examined by fluorescence microscopy. Our results demonstrated that mammalian cells produce HCO₄^– and boronate probes can evidence and distinguish it from H₂O₂ under biologically relevant concentrations of H₂O₂ and CO₂/HCO₃^–.