Ascorbate radical decay

The bimolecular decay of the ascorbate radical is much more complex than shown in the overall reaction (86). In fact, it is in equilibrium with a dimer [equilibrium (88), K 103 dm3 mol-1, kieverse 105 s"1] which either may react with a proton [reaction (89), k = 1010 dm3 mol-1 s"1] or with water [reaction (90), k 40 s"1] (Bielski et al. 1981). 

The ascorbate free radical decays by a strictly second-order disproportionation process to ascorbic acid and dehydroascorbic acid, independent of generation method. A product analysis (22) with l-ascorbate-l- C showed dehydroascorbate to be the only new product in an irradiated solution. 

Figure 2. Reaction rate, as a function of pH for the decay of ascorbate radicals at ambient temperature. Key Q, data from Ref. 23 , data from Ref. 22 and J, data from Ref. 14.

The first successful observation and characterization of the ascorbate free radical was carried out with ESR (14,15). A 1.7-G ESR doublet was reported and it was correctly concluded that the observed spectrum represented the anionic form (A ) of the radical. These measurements (14,15) showed that the enzyme-generated radical (horseradish peroxidase-hydrogen peroxide-ascorbate) was present as a free radical and decayed by second-order kinetics (see Figure 2). Recent experiments (16,17) have shown that ascorbate oxidase and dopamine-monooxygenase also generate unbound ascorbate radicals. 

In basic solutions ascorbate is apparently oxidized preferentially by the electron transfer process, which goes to completion in less than 2 fts after termination of the electron pulse (see Structure I). In nitrous-oxide-saturated acid solutions (pH 3.0-4.5), A and two other species which were shown to be OH-radical adducts were observed (37), thus confirming earlier observations (18,19,23, 25). The ascorbate radical anion was identified by its doublet of triplets spectrum that maintains its line position from pH 13 to 1. One OH-radical adduct (IV) shows a doublet, the lines of which start to shift below pH 3.0 it has a pK near 2.0, a decay period of about 100 fxs, and probably does not lead to formation of A". The other OH-radical adduct (II) is formed by addition of the OH radical to the C2 position its ESR parameters are = 24.4 0.0002 G and g == 2.0031 0.0002. Time growth studies suggest that this radical adduct converts to the ascorbate anion radical (III) with r 15 fxs, and accounts for 50% of the A signal intensity 40 fxS after termination of the electron pulse. The formation of the three radicals can be summarized as shown in Scheme 1. 

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 decay of A is complicated. A reaction scheme which has been suggested involves the combination of two ascorbate radical ions together to form an intermediate which then decomposes to form the products. It is necessary to propose such a mechanism in order to explain the dependence of the rate of the reaction on the ionic strength and the pH of the solution. The mechanism proposed is shown in equations (7)-(9). 

If the radiolytic species of interest has a fairly long lifetime, then a potentiostatic experiment will yield a Cottrell-like response after the pulse, because the faradaic current is controlled by diffusion, or possibly in part by the electrode kinetics. Data for this case are shown in Figure 18.3.4. The radiolytically generated radical of ascorbic acid decays on a millisecond time scale. Current transients following separate radiolytic pulses were recorded for different potentials, and samples were taken at a fixed delay time to produce the sampled-current voltammogram displayed in Figure 18.3.4. 

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. 

At 30°C in the absence of Arg, the ferrous-oxi complex transforms very slowly to the ferric state. In the presence of substrate and H4B, a new species with the 12-nm shifted Sorey band is detected. A decay of this species is accompanied by the formation ofN -hydroxy-L-arginine. Because the presence of HUB is necessary for these reactions, the main function of this compound is to be a reducing agent. This suggestion is supported by experiments on the stabilizing effect of ascorbic acid on the chemical stabilization of tetrahydropterin in the endothelial nitric oxide synthesis (Heller et al., 2001). At the same time, a significant increase in the half lifetime of H4B in solution is demonstrated. As is shown (Wei et al., 2001), a ferrous-dioxy intermediate in iNOS forms for 53 s 1 and then is transformed to the [S-Fe(IV)=0] state. The rate of the [S-Fe(IV)=0] decay is equal to the rate ofH4B radical formation and the rate of Arg hydroxylation. In contrast,... 

Ranganathan [4] enhanced the stability of MRI contrast imaging agents by incorporating ascorbic acid, (VI), to diminish oxidation of substituents from free radical reactions induced by radionuclide decay. 

By reaction with ascorbate to yield the monodehydroascorbate radical, which in turn can either be reduced to ascorbate or can undergo dis-mutation to yield dehydroascorbate and ascorbate (Section 13.4.7.1). In vitro, the formation of the tocopheroxyl radical can be demonstrated by the appearance of its characteristic absorbance peak, which normally has a decay time of 3 msec in the presence of ascorbate, the tocopheroxyl peak has a decay time of 10 /rsec, and its disappearance is accompanied by the appearance of the monodehydroascorbate peak. There is an integral membrane oxidoreductase that uses ascorbate as the preferred electron donor, linked either directly to reduction of tocopheroxyl radical or via an electron transport chain involving ubiquinone (see no. 4 below May, 1999). 

Ascorbate is a reactive reductant, but its free radical is relatively nonreactive (2) see Table I) and decays by disproportionation to ascorbic acid/ascorbate and dehydroascorbic acid ... 

Reduction Kinetics of the Type 3 Copper. Quantitative assessment of the complex kinetic behavior at 330 nm is diflScult. This diflSculty probably is partially caused by the small and varied contribution from the production and decay of the substrate radical species (63), a feature that is revealed when runs at at various wavelengths are compared. The decay rate of the radical, presumably decay by non-enzymatic dismutation, has a magnitude similar to the reduction rate of the type 3 copper. In the experiments with ascorbate as substrate. 

Excited-state porphyrins also are good oxidants. Photoreduction of uroporphyrin by EDTA and other electron donors [89] gives ESR spectra of the porphyrin radical anion, which has a low g value (2.002) and a broad ESR line (5 G) with no resolved hyperfine structure. The radical is transient and decays with second-order kinetics in aqueous solutions. Similar spectra have been reported from hematoporphyrin irradiated in the presence of thiols [88]. In addition, a thiol-derived radical was spin-trapped by MNP. The hematoporphyrin radical anion itself is not spin-trapped, but it will transfer an electron to MNP to form Bu NHO. With reducing agents other than thiols, radicals from the reducing agent (ascorbate, catechols, p-phenylenediamine) were detected directly [88]. 

The oxidation of ascorbic acid in certain reactions has given evidence of an intermediate with the properties of a free radical which could be formed by one-electron oxidation. Thus, the rate-limiting step of ascorbic acid oxidation by Fe + and H2O2 was this one-electron oxidation (G12). Such a radical has now been identified in hydrogen peroxide-ascorbic acid solutions at pH 4.8 by electron paramagnetic resonance spectroscopy. The free radical, commonly referred to as monodehydroascorbic acid, decayed in about 15 minutes at this acid pH. It was also formed during the enzymatic oxidation of ascorbic acid by peroxidase (Yl). The existence of the monodehydroascorbic acid radical makes possible very... 

Ascorbate is most likely required by hydroxylases to maintain iron or copper at the active enzyme site in the reduced form, since it is necessary for hydroxylation. The semidehydroascorbate radical is not very reactive (Bielski and Richter, 1975 Rose, 1989). It decays by disproportionation to ascorbate and dehydroascorbate (the latter subsequently degrades to oxalic acid and L-threonic acid), rather than acting as a reactive free radical. Reaction of ascorbic acid with (OH) is rapid and diffusion-dependent (K 7.2 x 10 -1.3 x lO o M- s- ) (Cabelli and Bielski, 1983). (O2) oxidizes ascorbic acid with a rate constant of 10 -10 M s (Bielski et al., 1985). Besides direct scavenging of radicals, ascorbic acid is known to have a number of physiological effects (Padh,... 

A laser flash photolysis study of the behaviour of the lowest excited triplet state and semiquinone radical anion of hypocrellin A (HA") suggests that, in the presence of substrates such as ascorbic acid and cysteine, formation and decay of (HA") occurs by electron transfer. The production of superoxide radical anion (O2") was also confirmed, and the conclusion is drawn from the experimental results that an electron transfer (Type I) mechanism may be important in the photodynamic interaction between HA and some biological substrates. Photoelectron transfer and hydrogen abstraction in the phenothiazine/p-benzoquinone system proceeds competitively, and a series of porphyrin quinones (5 = H,... 

CCI3O2 reacts with ascorbic and uric acid [71], as well as bilirubin [72] and glutathione [73] via electron transfer. However, with tryptophan and carotenoids another reaction also occurs, suggested to be radical addition [74, 75]. For the carotenoids the proposed adduct decays to yield more radical cation and for the carotenoid, astaxanthin, the radical cation is not formed initially but is formed solely through the proposed addition radical [75]. The one electron reduction potential of astaxanthin radical cation has been shown to be higher than several other carotenoids [76], so it may be that it is very close to that of CCI3O2 so that electron transfer is very slow. 

It is a significant fact that Chinoy, Singh and Laloraya (1974) have shown vitro that addition of DNA and RNA to an ascorbic acid solution with a trace of H2O2 accelerates both the formation of the free radical of AA-monodehydro-ascorbic acid and Its subsequent faster decay which ultimately leads to the formation of a complex between AA and the macromolecules Cref, Chapter 1). 

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