The kinetics of the reaction of nitrogen dioxide with iron(II)- and iron(III) cytochrome c.

The reactions of NO2 with both oxidized and reduced cytochrome c at pH 7.2 and 7.4, respectively, and with N-acetyltyrosine amide and N-acetyltryptophan amide at pH 7.3 were studied by pulse radiolysis at 23 °C. NO2 oxidizes N-acetyltyrosine amide and N-acetyltryptophan amide with rate constants of (3.1±0.3)×10(5) and (1.1±0.1)×10(6) M(-1) s(-1), respectively. With iron(III)cytochrome c, the reaction involves only its amino acids, because no changes in the visible spectrum of cytochrome c are observed. The second-order rate constant is (5.8±0.7)×10(6) M(-1) s(-1) at pH 7.2. NO2 oxidizes iron(II)cytochrome c with a second-order rate constant of (6.6±0.5)×10(7) M(-1) s(-1) at pH 7.4; formation of iron(III)cytochrome c is quantitative. Based on these rate constants, we propose that the reaction with iron(II)cytochrome c proceeds via a mechanism in which 90% of NO2 oxidizes the iron center directly-most probably via reaction at the solvent-accessible heme edge-whereas 10% oxidizes the amino acid residues to the corresponding radicals, which, in turn, oxidize iron(II). Iron(II)cytochrome c is also oxidized by peroxynitrite in the presence of CO2 to iron(III)cytochrome c, with a yield of ~60% relative to peroxynitrite. Our results indicate that, in vivo, NO2 will attack preferentially the reduced form of cytochrome c; protein damage is expected to be marginal, the consequence of formation of amino acid radicals on iron(III)cytochrome c.

Catalytic scavenging of peroxynitrite by lactoperoxidase in the absence and presence of bicarbonate.

The kinetics of the reaction of lactoperoxidase with peroxynitrite was studied under neutral and acidic pH. Lactoperoxidase catalyses peroxynitrite decay with the rate constant, k(c), increasing with decreasing pH. The values of k(c) obtained at pH 7.1, 6.1 and 5.1 are (1.9+/-0.1)x10(6), (5.0+/-0.1)x10(6) and (8.5+/-0.2)x10(6) M(-1)s(-1), respectively. This tendency means that peroxynitrous acid is the species involved in the reaction with the catalytic centre of lactoperoxidase. Lactoperoxidase is also able to scavenge peroxynitrite in the presence of bicarbonate with the rate constant identical, within experimental error, to that measured in the absence of bicarbonate. It is thus concluded that CO(3)-(.)/(.)NO(.2) radicals formed in the system do not inactivate LPO. The mechanism of the catalytic scavenging of peroxynitrite by LPO is proposed. The physiological relevance of this reaction is discussed.

Determination of hydroperoxides in aqueous solutions containing surfactants by the ferrous oxidation-xylenol orange method.

Some surfactants widely used as additives in food, pharmaceuticals, and cosmetic formulations are susceptible to peroxidation resulting in accumulation of hydroperoxides (HP). Our investigation was aimed to study the possible influence of different surfactants on the proportionality and reproducibility of the ferrous oxidation-xylenol orange method developed originally for the determination of hydroperoxides. We also attempted to apply this method to determine hydroperoxides produced radiolytically in surfactant molecules. From our preliminary studies we conclude that the method can be applied for determination of hydroperoxides in systems containing non-ionic or anionic surfactants provided careful calibration is performed for each surfactant.

Reactions of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) dianion, ABTS2-, with *OH, (SCN)2*-, and glycine or valine peroxyl radicals.

ABTS2-, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) dianion, was used as a reference to compare the reactivity of peroxyl radicals of two amino acids, glycine and valine, in aqueous solutions at natural pH. Peroxyl radicals were produced by pulse radiolysis and the product of their reaction with ABTS2- the ABTS*- radical was observed spectrophotometrically. Experimental kinetic traces were fitted using chemical simulation. The rate constants of reactions of glycine and valine peroxyl radicals with ABTS2- were (6.0+/-0.2)x10(6) and (1.3+/-0.1)x10(5) M-1.s-1, respectively. Moreover, it was found that only 60% of glycine radicals formed upon its reaction with *OH radicals reacted with molecular oxygen to yield peroxyl radicals. Comparison of experimental data with simulations of chemical reactions in irradiated ABTS and ABTS/NaSCN solutions showed that ABTS*- forms in the reaction with *OH with a yield of 43% and rate constant of (5.4+/-0.2)x10(9) M-1.s-1 and in the reaction with (SCN)2*- with a yield of 57% and rate constant of (8.0+/-0.2)x10(8) M-1.s-1.