HyPer7: High-level bacterial expression and kinetics showing significant oxidation by peroxynitrite and hypochlorous...

Genetically encoded probes of the HyPer family are regarded as specific for H₂O₂. However, this view may be premature due to the limited studies examining their reactivity with other biologically relevant oxidants under optimized conditions. Peroxynitrite is of particular interest due to its reactivity towards Cys residues, including the catalytic Cys of enzymes involved in cellular signaling. Here, we report the construction of a new plasmid, pET-15b-HyPer7, enabling high-level bacterial expression of HyPer7. The purified HyPer7 presented spectroscopic properties and sensitive response (F₅₀₀/F₄₀₀) to H₂O₂ (2-20 μM) consistent with previously reported data. In contrast, HyPer7 (1 μM) also responded to peroxynitrite and hypochlorous acid (10-50 μM), though less efficiently than to H₂O₂. In all cases, the predominant oxidation product was consistent with HyPer7 disulfide formation, as evidenced by UV-Vis and fluorescence spectra, along with reducing SDS-PAGE; peroxynitrite and hypochlorous acid also yielded secondary products. Stopped-flow kinetics of Hyper7 (1 μM) oxidation by various H₂O₂ (10-200 μM) or peroxynitrite concentrations (10-90 μM) allowed determination of second-order rate constants that differed by one order of magnitude, k_H2O2= (3.60 ± 0.50) x10⁵ M^-1 s^-1 and k_ONOOH= (2.15 ± 0.03) x 10⁴ M^-1 s^-1, in phosphate buffer, pH 7.4 at 25^οC. Hypochlorous acid kinetics proved more complex and require further studies; initial assessments suggest a rate constant in the order of 10⁶ M^-1 s^-1. Overall, our results indicate that HyPer7 is less specific to H₂O₂ than previously assumed. This conclusion likely extends to other members of the HyPer family due to the similar redox-sensitive domain.