HYDROGEN-PEROXIDE FORMATION BY REACTION OF PEROXYNITRITE WITH HEPES AND RELATED TERTIARY-AMINES - IMPLICATIONS FOR A GENERAL MECHANISM

Organic amine-based buffer compounds such as HEPES (Good's buffers) ar e commonly applied in experimental systems, including those where the biological effects of peroxynitrite are studied. In such studies 3-mor pholinosydnonimine N-ethylcarbamide (SIN-1), a compound that simultane ously releases nitric oxide ((NO)-N-.) and superoxide (O-2(.)) is ofte n used as a source for peroxynitrite. Whereas in mere phosphate buffer H2O2 formation from 1.5 mM SIN-1 was low (similar to 15 mu M), incuba tion of SIN-1 with Good's buffer compounds resulted in continuous H2O2 formation. After 2 h of incubation of 1.5 mM SIN-1 with 20 mM HEPES a bout 190 mu M H2O2 were formed. The same amount of H2O2 could be achie ved from 1.5 mM SIN-1 by action of superoxide dismutase in the absence of HEPES. The increased H2O2 level, however, could not be related to a superoxide dismutase or to a NO scavenger activity of HEPES, On the other hand, SIN-1-mediated oxidation of both dihydrorhodamine 123 and deoxyribose as well as peroxynitrite-dependent nitration of p-hydroxyp henylacetic acid were strongly inhibited by 20 mM HEPES, Furthermore, the peroxynitrite scavenger tryptophan significantly reduced H2O2 form ation from SIN-1-HEPES interactions. These observations suggest that p eroxynitrite is the initiator for the enhanced formation of H2O2. Like wise, authentic peroxynitrite (1 mar) also induced the formation of bo th O-2(.) and H2O2 upon addition to HEPES (400 mM)-containing solution s in a pH (4.5-7.5)-dependent manner. In accordance with previous repo rts it was found that at pH greater than or equal to 5 oxygen is relea sed in the decay of peroxynitrite, As a consequence, peroxynitrite(1 m M)-induced H,O, formation (similar to 80 mu M at pH 7.5) also occurred under hypoxic conditions. In the presence of bicarbonate/carbon dioxi de (20 mM/5%) the production of H,O, from the reaction of HEPES with p eroxynitrite was even further stimulated. Addition of SIN-1 or authent ic peroxynitrite to solutions of Good's buffers resulted in the format ion of piperazine-derived radical cations as detected by ESR spectrosc opy. These findings suggest a mechanism for H2O2 formation in which pe roxynitrite (or any strong oxidant derived from it) initially oxidizes the tertiary amine buffer compounds in a one-electron step. Subsequen t deprotonation and reaction of the intermediate alpha-amino alkyl rad icals with molecular oxygen leads to the formation of O-2(.), from whi ch H2O2 is produced by dismutation, Hence, HEPES and similar organic b uffers should be avoided in studies of oxidative compounds. Furthermor e, this mechanism of H2O2 formation must be regarded to be a rather ge neral one for biological systems where sufficiently strong oxidants ma y interact with various biologically relevant amino-type molecules, su ch as ATP, creatine, or nucleic acids.