235 - Influence of pH on the reactions of heme peroxidase-derived oxidants with R19S

Abstract

Oxidant production by heme peroxidases (such as myeloperoxidase [MPO], lactoperoxidase, eosinophil peroxidase and peroxidasin) impacts host defense and macromolecular structure and has the potential to cause tissue damage. Marked increases in phagocyte MPO, capable of producing highly reactive hypochlorous acid (HOCl), have been noted in multiple chronic inflammatory diseases. However, measuring MPO-derived oxidants at the site of production in vivo is technically challenging. The rhodamine-based probe R19S, first described by X. Chen et al (Chem Commun 2011; 47:4373-4375), becomes fluorescent R19 after reaction with HOCl but does not react with superoxide, hydrogen peroxide or several other reactive species. Thus, R19S may be useful to study MPO activity in situ. We sought to characterize the potential responses of R19S to other heme peroxidase-derived oxidants to establish whether a fluorescent response is solely indicative of HOCl or may include others. We analyzed responses of R19S to HOCl (positive control), hydrogen peroxide (negative control), hypobromous acid (HOBr), hypothiocyanous acid (HOSCN), hypoiodous acid (HOI) and taurine chloramine (TauCl) at pH 5.5 and 7.5. A similar study was recently published by A.M. Albrett et al (J Biol Chem 2018; ahead of print). Our results show strong agreement with their study at neutral pH (with a response hierarchy of HOI > HOCl > HOBr > HOSCN), although the overall responses of HOBr and HOSCN relative to HOCl were greater. At pH 5.5, we observed an altered hierarchy, with HOBr and HOI responses diminished (mean of 14.0±0.5% and 65.1±1.0% of HOCl, respectively, vs. 83.2±2.3% and 165.8±6.4% at pH 7.5), while HOSCN response was increased (93.3±5.1% of HOCl, vs. 37.6±0.2% at pH 7.5). Although TauCl did not cause fluorescence at neutral pH, modest response was detectible at acidic pH (9.8±0.2% of HOCl). These data illustrate that the environment of R19S determines its responsiveness to peroxidase-derived oxidants and should be considered when interpreting results.