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J Biol Chem, Vol. 273, Issue 21, 12716-12724, May 22, 1998

Hydrogen Peroxide Formation by Reaction of Peroxynitrite with HEPES and Related Tertiary Amines
IMPLICATIONS FOR A GENERAL MECHANISM

Michael Kirsch, Elena E. Lomonosova, Hans-Gert Korth^§, Reiner Sustmann^§, and Herbert de Groot

From the  Institut für Physiologische Chemie, Universitätsklinikum, Hufelandstrasse 55, D-45122 Essen and ^§ Institut für Organische Chemie, Universität-GH Essen, Universitätsstrasse 5, D-45117 Essen, Germany

Organic amine-based buffer compounds such as HEPES (Good's buffers) are commonly applied in experimental systems, including those where the biological effects of peroxynitrite are studied. In such studies 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), a compound that simultaneously releases nitric oxide (^·NO) and superoxide (O₂), is often used as a source for peroxynitrite. Whereas in mere phosphate buffer H₂O₂ formation from 1.5 mM SIN-1 was low (~15 µM), incubation 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 about 190 µM H₂O₂ were formed. The same amount of H₂O₂ could be achieved 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-hydroxyphenylacetic acid were strongly inhibited by 20 mM HEPES. Furthermore, the peroxynitrite scavenger tryptophan significantly reduced H₂O₂ formation from SIN-1-HEPES interactions. These observations suggest that peroxynitrite is the initiator for the enhanced formation of H₂O₂. Likewise, authentic peroxynitrite (1 mM) also induced the formation of both O₂ and H₂O₂ upon addition to HEPES (400 mM)-containing solutions in a pH (4.5-7.5)-dependent manner. In accordance with previous reports it was found that at pH 5 oxygen is released in the decay of peroxynitrite. As a consequence, peroxynitrite(1 mM)-induced H₂O₂ formation (~80 µM at pH 7.5) also occurred under hypoxic conditions. In the presence of bicarbonate/carbon dioxide (20 mM/5%) the production of H₂O₂ from the reaction of HEPES with peroxynitrite was even further stimulated. Addition of SIN-1 or authentic peroxynitrite to solutions of Good's buffers resulted in the formation of piperazine-derived radical cations as detected by ESR spectroscopy. These findings suggest a mechanism for H₂O₂ formation in which peroxynitrite (or any strong oxidant derived from it) initially oxidizes the tertiary amine buffer compounds in a one-electron step. Subsequent deprotonation and reaction of the intermediate -amino alkyl radicals with molecular oxygen leads to the formation of O₂, from which H₂O₂ is produced by dismutation. Hence, HEPES and similar organic buffers should be avoided in studies of oxidative compounds. Furthermore, this mechanism of H₂O₂ formation must be regarded to be a rather general one for biological systems where sufficiently strong oxidants may interact with various biologically relevant amino-type molecules, such as ATP, creatine, or nucleic acids.

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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