2-2--azino-di-(3-ethylbenzothiazoline)-6-sulfonic-acid and peroxynitric-acid

Inhibition of oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by free radicals generated by decomposition of 2,2'-azobis(2-amidopropane) (ABAP) by antioxidants and biological material was studied. A correlation was found between the ability of various substances to delay the onset of ABTS oxidation and their rapid reduction of the ABTS+* cation radical, and between the ability to reduce the maximal rate of ABTS oxidation and slow reduction of ABTS+*. The length of the lag period of ABTS oxidation was found to be independent of ABTS concentration. Similar decrease of peroxynitrite-induced ABTS+* formation by antioxidants was observed when the antioxidants were added before and after peroxynitrite. All these findings indicate that the main effect of antioxidants in this system is reduction of ABTS+* and not prevention of its formation. Reduction of oxidation products rather than inhibition of their formation may be the predominant mode of action of antioxidants in various assays of antioxidant activity.

Topics: Amidines; Antioxidants; Benzothiazoles; Nitrates; Oxidants; Oxidation-Reduction; Sulfonic Acids

Nitric oxide reacts rapidly with superoxide to give the strongly oxidizing peroxynitrite anion (ONOO-), which undergoes spontaneous first-order decomposition when protonated. The oxidative chemistry of peroxynitrite (ONOO-) is highly pH-dependent. At acidic pH, peroxynitrous acid (ONOOH) oxidizes dimethylsulfoxide to formaldehyde and 2,2'-azino-bis-(3-ethyl-1,2- dihydrobenzothiazoline 6-sulfonate) (ABTS) to the greenish-colored ABTS+ radical cation. The product yield from dimethylsulfoxide and ABTS decreased at more alkaline pH with apparent pK(a)s of 7.9 and 8.2, respectively. Decreasing yield with increasing pH could not be explained by the oxidation of either formaldehyde or ABTS+ by peroxynitrite. In the presence of 50 mM dimethylsulfoxide, nitrogen dioxide was formed in approximately equimolar amounts to the other reaction product, formaldehyde. The yield of nitrogen dioxide also decreased with an apparent pK(a) of 8.0. We propose that the complex oxidative chemistry of peroxynitrite is controlled by the pH-dependent isomerization of the relatively stable cis-configuration (predominant at high pH) to the trans-configuration. Trans-peroxynitrous acid can form a vibrationally excited intermediate capable of reacting like hydroxyl radical. The vibrationally excited intermediate can also directly rearrange to nitric acid, reducing the apparent hydroxyl radical yield to less than 30%. The loss of hydroxyl radical-like reactivity can be explained on the basis of ionization of trans-peroxynitrous acid to the trans-anion, which in turn undergoes internal rearrangement to nitrate without forming a strong oxidant.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Benzothiazoles; Dimethyl Sulfoxide; Formaldehyde; Free Radical Scavengers; Free Radicals; Hydrogen-Ion Concentration; Hydroxyl Radical; In Vitro Techniques; Isomerism; Nitrates; Oxidation-Reduction; Sulfonic Acids

A recent study reports discordant results for maximal free-radical yields from the decomposition of peroxynitrous acid using different assay reagents and a puzzling decrease (to zero) of the radical yields with increasing pH. An assay method using 2,2,'azino-bis(3-ethyl-benzthiazoline sulphonate) (ABTS) has been tested using stopped-flow kinetic studies, and the results imply that the assay is satisfactory when [HOONO] much greater than [-OONO] but that reactions involving peroxynitrite anion interfere at higher pH. Evidence is presented that peroxynitrite was an interfering species in the earlier studies.

Topics: Benzothiazoles; Free Radicals; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Nitrates; Nitric Oxide; Sulfonic Acids; Superoxides