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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-morpholinosydnonimineN-ethylcarbamide (SIN-1), a compound that simultaneously releases nitric oxide (ċNO) and superoxide (), is often used as a source for peroxynitrite. Whereas in mere phosphate buffer H2O2 formation from 1.5 mmSIN-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 ofp-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 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 α-amino alkyl radicals with molecular oxygen leads to the formation of 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 H2O2formation 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.
The term “peroxynitrite” is commonly used to describe the equilibrium mixture of oxoperoxonitrate(1−) (ONOO−) and its conjugated acid, hydrogen oxoperoxonitrate(1−) (peroxynitrous acid, ONOOH). Peroxynitrite is a strong oxidant formed in the diffusion-controlled reaction of superoxide () and nitric oxide (nitrogen monoxide, ċNO) (k = 3.9–6.7 × 109m−1s−1) (
In obvious contradiction to the assumption of a central role of ONOO−/ONOOH in cell injuring processes, almost complete protection from SIN-1 cytotoxicity in experiments with rat liver endothelial cells and Fu5 rat hepatoma cells was provided by catalase but not by SOD (
), these results strongly suggest a participation of H2O2 in SIN-1-mediated cytotoxicity rather than a participation of ONOO−/ONOOH. Indeed, formation of H2O2 from SIN-1 was observed under certain experimental conditions (
) have recently demonstrated that the formation of H2O2, and consequently the protection exerted by catalase, decisively depends on the presence of the organic buffer compound HEPES in the incubation medium. In the absence of this “Good's buffer” neither H2O2 was formed nor was catalase protective. The question, however, how HEPES and similar Good's buffers (
) mediate the formation of H2O2 from SIN-1 remained open. The present study aims at the elucidation of the underlying chemical mechanism.
Reactions between authentic or in situ generated peroxynitrite and HEPES or PIPES result in the formation of HEPES- and PIPES-derived radical cation species (R1 and R2) (Fig. 8) as shown by ESR spectroscopy (Fig. 7). A reasonable mechanism for the formation of R1 and R2 is displayed in Fig. 11, where [Ox] stands for the action of any sufficiently strong oxidant in the system,e.g. peroxynitrite and peroxynitrite-derived oxidants formed in the absence and presence of carbon dioxide, without specifying the actual reactive species.
Fig. 11 also provides the starting point for the interpretation of formation in our system. It is common knowledge that carbon-centered radicals react rapidly (k ≈2 × 109m−1 s−1) with molecular oxygen to give peroxyl radicals (
) in the case of electron-rich alkyl radicals, i.e. those carrying electron-donating substituents, the corresponding peroxyl radicals are highly unstable, decomposing rapidly (k≈104-109m−1s−1) to a cationic species and (
By analogy, we propose that a similar mechanism is operative in the production of from Good's buffers by attack of authentic or SIN-1-generated peroxynitrite (Fig.12). Thus, it would appear that the rate of production is governed by the first two steps (electron transfer and deprotonation) of the reaction sequence. On the other hand, R4 may also be generated directly from the parent compound by hydrogen abstraction by suitably reactive radicalsXċ, e.g. hydroxyl or peroxyl radicals. In line with this assumption, formation of R1 and R4has been postulated in the autoxidation of DNA/Cu2+/H2O2 systems in the presence of HEPES and PIPES (
Further consequences arise from the foregoing. First, /H2O2 production is expected to be pH-dependent, decreasing with decreasing pH and reflecting the respective pKa of the organic buffer compound. This hypothesis was confirmed by the data of Fig. 10 and Table IV, in agreement with the requirement of an unshared electron pair at nitrogen for the oxidation process. Second, the efficiency of /H2O2 production among the various buffer compounds should be somehow affected by the structure and properties of the side chains attached to the piperazine rings. As amino-derived radical cations are strong electrophiles and are normally rapidly quenched in aqueous solution (
), one might hypothesize that the negatively charged sulfonic acid side chain(s) exert some “protective” effect on the cationic radical center inR1, thereby inhibiting the rate of deprotonation. This would agree with the observations that the monosulfonylated piperazines HEPES and EPPS gave higher yields of H2O2 (Tables Iand IV) than the bis-sulfonylated POPSO and PIPES, but, vice versa, the HEPES-derived radical showed a much lower ESR signal intensity (steady-state concentration) than the PIPES-derived one.
There is no contradiction in the fact that the ESR spectra of radical cations R1 and R2 have been detected although their reaction with oxygen is extremely fast. What we detected by ESR is just the small “excess” amount of R1 andR2 after all of the dissolved oxygen has been consumed. This explains the sometimes observed short term growth of the ESR signals immediately after mixing of the reactants.
The results presented here clearly demonstrate that the interaction of peroxynitrite and piperazine-type buffers, regardless of the presence of HCO3−/CO2, may lead to serious consequences concerning the investigation of peroxynitrite-driven actions under physiological conditions,e.g. necrosis, apoptosis, inhibition of enzymes, formation of metabolites, etc. There are several reports in the literature in which similar effects of the interaction of HEPES with strong oxidants other than peroxynitrite are mentioned, although no satisfying explanation has been given. For example, vanadyl induces hemolysis of vitamin E-deficient erythrocytes in HEPES buffer but not in phosphate buffer (
). In conclusion, it must be emphasized that /H2O2 formation according to the above mechanism (Fig. 12) is not restricted to peroxynitrite as an oxidant and not to piperazine buffer compounds as targets but should be regarded as a general pathway for compounds from which electron-rich alkyl radicals (preferably α-amino or α,α-dialkoxyl radicals) can be generated. Accordingly, H2O2 formation in the range of 75 μm has been found in the reaction of peroxynitrite (1 mm) with other tertiary amines (400 mm), viz. triethylamine and triethanolamine.
M. Kirsch, H. G. Korth, R. Sustmann, and H. de Groot, unpublished observations.
Generally speaking, any oxidant strong enough to oxidize certain tertiary amines in a one-electron step would be able to initiate the above reaction sequence. Furthermore, any other reaction, preferably hydrogen abstraction, that generates α-amino alkyl or α,α-dialkoxyl radicals would induce the same process. Because tertiary amine groups as targets for strong oxidants are present in a variety of biological molecules, e.g. in ATP, creatine, or nucleic acids, we propose that Fig. 12 for /H2O2 production must be considered to be a general mechanism under in vivoconditions. This certainly sheds a new light on a number of studies aimed at the pathophysiological effects of various oxidative species, as actually hydrogen peroxide might have been the true operating agent.
We thank Angela Wensing for excellent technical assistance.