Bechara, E; Massari, J; Tokikawa, R; Assuncao, NA; Zanolli, L; Tavares, MFM
Methylglyoxal (MG) is an α-oxoaldehyde putatively produced in excess by triose phosphates, aminoacetone and acetone in some disorders, particularly in diabetes. in turn, ONOO– is a potent oxidant and nucleophile for med in vivo by a diffusion controlled reaction between O2•− and •NO radicals. Here, we study the nucleophilic addition of ONOO– to MG in normally aerated phosphate buffer yielding a nitrosoperoxoadduct, whose homolysis leads to acetyl radical and ultimately acetate ions. Using stopped-flow UV spectrophotometry, the rate of ONOO– decay in the presence of MG (k2 = 1.0 × 105 M-1s-1), pH 7.2, at 25ºC, was found to be much faster than that reported for monocarbonyl substrates (k2 < 103 M-1s-1) as well as for diacetyl (k2= 1.0 × 104 M-1s-1) and CO2 (k2 = 5.8 × 104 M-1s-1) (Massari et al., CRT 2008;21:879). the reaction displays a pH profile with a shoulder at pH 7.2, coincident with the pKa values of both ONOOH and H2PO4–, which is in accordance with the hypothesis of phosphate-catalyzed nucleophilic addition of ONOO– to MG. the ascendent portion of the curve above pH 8 is explainable by HPO4_2–catalyzed dehydration of MG. H2O to the free, reactive form of MG (Thornalley et al., Biochem. Pharmacol. 2000;60:55). EPR spin trapping studies with MNP revealed for mation of an adduct attributable to MNP-CH3CO• radical (aN = 0.83 mT), whose triplet signal intensity responds to both [ONOO–] and [MG]. Trapping with DBNBS gave a signal assignable to a methyl radical adduct (aN = 1.41 mT; aH = 1.35 mT; aH(m) = 0.08 mT). The acetyl radical adduct could also be observed by replacing ONOO– for H2O2, although at much lower yields. the extent of O2 uptake by the ONOO–/MG system is concentration-dependent. Stoichiometric amounts of for mate and acetate were detected by capillary electrophoresis in the spent reaction mixture. Methylglyoxalate, possibly for med by direct MG oxidation, was absent. These data support our hypothesis that DNA and protein acetylation may take place in vivo by a radical route under pathological conditions where ONOO–/H2O2 and α-dicarbonyls accumulate, as seems to be the case of inflammatory processes. Support: FAPESP, CNPq, Milenio Redoxoma.
