The
nitroxyl (HNO) donor
Angeli's salt (Na(2)N(2)O(3); 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, H(2)O(2) 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 H(2)O(2) 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 H(2)O(2). 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 O(2) and HNO produced by AS.