Reaction of Peroxynitrite with HEPES or MOPS Results in the Formation of Nitric Oxide Donors

We investigated the effects of organic buffers on the NO-like biological activities of ONOO−. In HEPES buffer (50 mM), ONOO− (1 mM) induced a 20-fold increase in endothelial cGMP accumulation and the effect was comparable to that elicited by a maximally active concentration of the NO donor DEA/NO. ONOO− produced a 12-fold increase of cGMP in MOPS buffer (50 mM), but was virtually inactive in phosphate buffer (50 mM). Electrochemical detection of NO showed that the biological effects of ONOO− in HEPES or MOPS were due to accumulation of compounds that released NO in the presence of copper ions. CuCl2-induced formation of NO was completely blocked by the Cu(I) chelator neocuproine but unaffected by the Cu(II) chelator cuprizone, pointing to a Cu(I)-catalyzed decomposition pathway. Formation of NO from ONOO− was not detectable in phosphate buffer, in agreement with the lack of effect of ONOO− on cGMP accumulation in this buffer. These data demonstrate that certain buffer components present in cell culture media may yield artificial results in experiments with authentic ONOO−.