Adrenaline autoxidation

The oxidation of catecholamines like epinephrine has been widely used as source for superoxide dismutase assays. Upon oxidation the catecholamines are transformed to the coloured product adrenochrome. The rate of oxidation by superoxide is inhibited in the presence of superoxide dismutases Likewise the autoxidation of catecholamines at alkaline pH-values is diminished Intriguingly, low molecular mass copper complexes which display superoxide dismutase activity accelerate the autoxidation Therefore, the interaction between superoxide and catecholamines and its inhibition by SOD is thought not to be a simple chemical reactionRecently, this reaction was investigated in more detail Whilst adrenalin autoxidation is very specifically inhibited by SOD, the reaction with other catecholamines like noradrenalin or dihydroxyphenylalanine, having no free amino group, is much less specific. Only 20 % inhibition by CujZnjSuperoxide dismutase are observed. The autoxidation reaction itself is very complex (Scheme 2) and still not fully understood. 

The experimental observations were interpreted by assuming that the redox cycle starts with the formation of a complex between the catalyst and the substrate. This species undergoes intramolecular two-electron transfer and produces vanadium(II) and the quinone form of adrenaline. The organic intermediate rearranges into leucoadrenochrome which is oxidized to the final product also in a two-electron redox step. The +2 oxidation state of vanadium is stabilized by complex formation with the substrate. Subsequent reactions include the autoxidation of the V(II) complex to the product as well as the formation of aVOV4+ intermediate which is reoxidized to V02+ by dioxygen. These reactions also produce H2O2. The model also takes into account the rapidly established equilibria between different vanadium-substrate complexes which react with 02 at different rates. The concentration and pH dependencies of the reaction rate provided evidence for the formation of a V(C-RH)3 complex in which the formal oxidation state of vanadium is +4. 

Superoxide can also be produced by other enzymes, such as the range of flavin oxidases located in peroxisomes, and by oxidation of certain compounds including ascorbic acid, thiols, and adrenaline in the presence of transition metal ions. The autoxidation of reduced transition metal can also generate the superoxide... 

The detection limit of the autoxidation assay is 0.5 nM superoxide dismutase The oxidation of adrenalin is followed at 480 nm spectrometrically. 850 pi 100 mM carbonate buffer, pH 10 and 100 pi water or sample are mixed. The reaction is started with 50 pi catecholamine stabilized at pH 2. AA/min is followed. Due to its convenience, this method can be employed in homogenates and other biological samples However, the assay can interfere with reduced glutathione, causing an... 

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