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While has been known since the late sixties that superoxide is formed in vivo, the discovery of nitrogen monoxide, also known as nitric oxide, production by nitric oxide syntheses is relatively recent. Nitrogen monoxide and superoxide react to form peroxynitrile, O=NOO- . Subsequent research has shown that this isomer of nitrate is harmful: thiols, such as glutathione, are oxidized, tyrosines are nitrated and lipid peroxidation is initiated. Peroxynitrite reacts in three different ways, as follows from the kinetics with various small molecule.
(1) The rate of reaction of peroxynitrite with phenolic compounds does not depend on the concentration of the phenolic compound. The rate is the same as that of the rate of isomerization of nitrate.
(2) The rate of reaction of peroxynitrite with compounds like ascorbate, tryptophan and thiols depends on the concentration of both reactants. Such reactions could be used to scavenge peroxynitrite.
(3) Peroxynitrite binds to a metalion and forms a nitrating species.
In order to study peroxinitrite we developed a new synthesis. Improvements to the biomimetic synthesis of peroxynitrite with solid potassium superoxide and gaseous nitrogen monoxide results in higher peroxynitrite to nitrite yields than in most other syntheses.
Recent research in our group has shown that the rate of reaction of nitrogen monoxide with superoxide is faster than previously thought. Flash photolysis of alkaline peroxynitrite solutions results in the formation of nitrogen monoxide and superoxide. From the rate of recombination it is concluded that the rate constant of the reaction of nitrogen monoxide with superoxide is (1.9±0.2)x 1010 /Ms . This value implies that near activated macrophages the formation of peroxynitrite cannot be prevented by the enzyme superoxide dismutase. The pKa of hydrogen oxoperoxonitrate is dependent on the medium. With the stopped-flow technique a value of 6.5 is found at millimolar phosphate concentrations while at 0.5 M phosphate the value is 7.5. Thus, it is not known what the pKa in vivo, and therefore it is known what the relative concentrations are of the protonated form and the anion. It was further found that the kinetics of isomerization do not follow first-order kinetics when the pH is larger than the pKa, combined with a total peroxynitrite and peroxynitrous acid concentrations that exceeds of 0.1 mM. An adduct between ONOO- and ONOOH is formed with a stability of 1.0±0.1 x 10**4 M. Such adducts may also be formed with biomolecules. We also attempted to oxidize peroxynitrite. Pulse radiolysis of alkaline peroxynitrite solutions indicate that the hydroxyl radical reacts with ONOO- to form [(HO)ONOO]²- with the rate constant of 5.8x10**9/Ms . This radical absorbs with a maximum at 420 nm (e=1.8x10**3/M cm ), and decays by second -order kinetics , k = 3.4x10**6/Ms .
supported by EHT and the Swiss Nationalfonds