The nitroxyl (HNO) donor Angeli's salt (Na2N2O3; AS) is cytotoxic in vitro, inducing double strand DNA breaks and base oxidation, yet may have pharmacological application in the treatment of cardiovascular disease. The chemical profiles of AS and synthetic peroxynitrite (ONOO-) in aerobic solution were recently compared, and AS was found to form a distinct reactive intermediate. However, similarities in the chemical behavior of the reactive nitrogen oxide species (RNOS) were apparent under certain conditions. Buffer composition was found to have a significant and unexpected impact on the observed chemistry of RNOS, and varied buffer conditions were utilized to further distinguish the chemical profiles elicited by the RNOS donors AS and synthetic ONOO-. Addition of HEPES to the assay buffer significantly quenched oxidation of dihydrorhodamine (DHR), hydroxylation of benzoic acid (BA), and DNA damage by both AS and ONOO-, and oxidation and nitration of hydroxyphenylacetic acid by ONOO-. Additionally, H2O2 was produced in a concentration-dependent manner from the interaction of HEPES with both the donor intermediates. Interestingly, clonogenic survival was not affected by HEPES, indicating that H2O2 is not a contributing factor to in vitro cytotoxicity of AS. Variation in RNOS reactivity was dramatic with significantly higher relative affinity for the AS intermediate toward DHR, BA, DNA, and HEPES and increased production of H2O2. Further, AS reacted to a significantly greater extent with the unprotonated amine form of HEPES while the interaction of ONOO- with HEPES was pH-independent. Addition of bicarbonate only altered ONOO- chemistry. This study emphasizes the importance of buffer composition on chemical outcome and thus on interpretation and provides further evidence that ONOO- is not an intermediate formed between the reaction of O2 and HNO produced by AS.
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