Steady state ascorbate radicals

Equilibrium studies under anaerobic conditions confirmed that [Cu(HA)]+ is the major species in the Cu(II)-ascorbic acid system. However, the existence of minor polymeric, presumably dimeric, species could also be proven. This lends support to the above kinetic model. Provided that the catalytically active complex is the dimer produced in reaction (26), the chain reaction is initiated by the formation and subsequent decomposition of [Cu2(HA)2(02)]2+ into [CuA(02H)] and A -. The chain carrier is the semi-quinone radical which is consumed and regenerated in the propagation steps, Eqs. (29) and (30). The chain is terminated in Eq. (31). Applying the steady-state approximation to the concentrations of the radicals, yields a rate law which is fully consistent with the experimental observations ... 

According to ESR measurements, the semiquinone radical forms at nM concentration levels and its steady-state concentration was reported to increase by increasing the total concentration of ascorbic acid. The kinetic role of O was confirmed by the inhibitory effect of... 

In the oxidation of ascorbate by Oj catalysed by ascorbate oxidase, the formation of the monodehydroascorbate free radical was demonstrated by EPR spectroscopy in a flow cell. A steady state was usually reached within 50 ms. The production of the free radical was also followed by the reduction of Fe(in)-cytochrome c. Thus the oxidation of ascorbate occurs in a one-electron step The formation of the monodehydroascorbate free radical was also measured directly by spectrophotometry at 360 nm, where the free radical shows an absorption maximum... 

Figure 13.10. Schematic representation of the oxide dissolution processes [exemplified for Fe(III) (hydr)oxides] by acids (H ions), ligands (example oxalate), and reductants (example ascorbate). In each case a surface complex (proton complex, oxalato and ascorbato surface complex) is formed, which influences the bonds of the central Fe ions to O and OH on the surface of the crystalline lattice, in such a way that a slow detachment of a Fe(III) aquo or a ligand complex [in case of reduction an Fe(ll) complex] becomes possible. In each case the original surface structure is reconstituted, so that the dissolution continues (steady-state condition). In the redox reaction with Fe(III), the ascorbate is oxidized to the ascorbate radical A . The principle of proton-promoted and ligand-promoted dissolution is also valid for the dissolution (weathering) of Al-silicate minerals. The structural formulas given are schematic and simplified they should indicate that Fe(III) in the solid phase can be bridged by O and OH.

Initial experiments involved the use of hydroquinones, ascorbate and dihydroxy fumarate as substrates for the enzyme [120], Radicals (e.g., 5 and 6) were detected under steady-state conditions using a flow system to minimize substrate depletion. The narrow line spectra (Fig. 6a,b) of the radical anions are identical to those generated in chemical systems, indicating that the radicals are free in solution rather than associated with the enzyme. If it is assumed that the reaction proceeds by one-electron oxidation of the substrate and that the product radicals decay by self-reaction (e.g., disproportionation), kinetic analysis predicts that the steady-state radical... 

The term auto-oxidation is often used to refer to the oxidation of l-ascorbic acid by molecular oxygen in aqueous solution in the absence of any catalysts. The many studies on this topic still have not produced an entirely satisfactory mechanism for the electron transfer from ascorbate to the dioxygen molecule. E.s.r. studies have shown that when ascorbate reacts with dioxygen in aqueous solution in the pH range 6.6-9.6, a steady-state concentration of ascorbate radicals is produced. There is, however, little direct evidence for the formation of H2O2 in this reaction. Data from the inhibition of the reaction by certain enzymes, notably superoxide dismutase, suggest that H2O2 is produced in the process. 

With the exception of a study carried out with a partially characterized multicopper oxidase isolated from tea leaves (85), there has been very little detailed work concerned with the steady state kinetic behavior of laccases. Early work on the transient kinetics indicated, however, that (1) enzyme bound Cu + was reduced by substrate and reoxidized by O2, and (2) substrate was oxidized in one-electron steps to give an intermediate free radical in the case of the two electron donating substrates such as quinol and ascorbic acid. The evidence obtained suggested that free radicals decayed via a non-enzymatic disproportionation reaction rather than by a further reduction of the enzyme (86—88). In the case of substrates such as ferrocyanide only one electron can be donated to the enzyme from each substrate molecule. It was clear then that the enzjmie was acting to couple the one-electron oxidation of substrate to the four-electron reduction of oxygen via redox cycles involving Cu. 

Roginski, V. A., and 8tegmann, H. B., 1993, Kinetics of the reaction between ascorbate and free radical from vitamin E as studied by E8R steady-state method, Chem. Phys. Lipids 65 103-112. 

Ascorbyl radical can be measured in a steady-state concentration in fresh milk. Oxidation of ascorbate by lactoperoxidase has been proposed to be the source of this radical, based on the increase in ESR signal upon an increase in the concenfra-tion of HjOjand the decrease in signal upon addition of azide (a lactoperoxidase inhibitor) (8). However, the radical may also stem from autoxidation of ascorbic acid in die presence of transition metals (9). 

Dehydroascorbate is the dominant oxidation product at pH 7.4, confirming the arguments based on simple algebra discussed above. The uncertainties in the kinetics of some reactions in the model should not change this conclusion although the predictions of the levels of oxysulphur products are obviously subject to a great deal of uncertainty, they must be in total only a fraction of the oxidized ascorbate. From the steady-state concentrations of radical intermediates listed (note the numerical values tabulated are all micromolar, so the concentra-... 

The reactions of H atoms and OH radicals with ascorbic acid have been investigated by pulse radiolysis FT EPR. The rate constant of the addition of H atoms to ascorbic acid at pH 1 was directly determined by the change of linewidth of the low-field line of the H atom in the presence of ascorbic acid (fcuadd = 1.3 X 10 M s ). In basic solution the addition ofthe OH radical results in two ascorbic acid radicals, the radical-anion and the OH adduct at position 3, corresponding to steady-state EPR measurements by Larolf et The kinetics... 

The resemblance between the model hydroxylating system of Uden-friend and the phenolase plus ascorbic acid system is real, as in both cases probably free radicals and certainly hydrogen peroxide are formed. The steady state concentration of the hydrogen peroxide is much higher in the former than in the latter system, and this difference alone can of course explain the fact that the catalase inhibits the hydroxylation... 

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