Hydrogen peroxide, ascorbate peroxidase

Peroxidases are found in milk and in leukocytes, platelets, and other tissues involved in eicosanoid metabolism (Chapter 23). The prosthetic group is protoheme. In the reaction catalyzed by peroxidase, hydrogen peroxide is reduced at the expense of several substances that will act as electron acceptors, such as ascorbate, quinones, and cytochrome c. The reaction catalyzed by peroxidase is complex, but the overall reaction is as follows ... 

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. 

Floyd. R. A., Soong. L. M., and Culver, P. L. Horseradish peroxidase/ hydrogen peroxide-catalyzed oxidation of the carcinogen M-hydroxy-Af-acetyl-2-aminofluorene as affected by cyanide and ascorbate. Cancer Res.. 36 1510-1519.1976. 

Among the donors known to be oxidized by the peroxidase-hydrogen peroxide system are mono- and polyphenols, aminophenols, diamines, leuco bases of several dyes, indophenols, amino acids with reactive groups, ascorbic acid, etc. Extensive lists of donors which give colored oxidation products can be found in the papers of Kastle (202) and Josl (198). The oxidation of amino acids and proteins is discussed by Sizer (321) and reports on other donor substances are found scattered over numerous publications. 

Fig. 1. This illustrates the zero-order reaction kinetics of the oxidation of ascorbic acid by horseradish peroxidase-hydrogen peroxide complex. The disappearance of hydrogen peroxide upon the addition of 3 mM ascorbic acid is recorded by means of the platinum microelectrode (Expt. 574e).

Britton Chance (16), using a modification of the Hartridge-Roughton flow method, studied the kinetics of the formation and dissociation of a primary peroxidase-hydrogen peroxide compound, and its reaction with acceptors like ascorbic acid and malachite green ... 

Takahama, U. Oniki, T. A peroxidase/phenolics/ascorbate system can scavenge hydrogen peroxide in plant cells. Physiol. Plantarum 1997, 101, 845-852. 

Hermann (2000) described a rapid automated method involving generation of a known amount of free radicals and the detection of the excess by photochemiluminescence. Test kits are available for determination of total water-soluble antioxidants, fat-soluble antioxidants and ascorbic acid. A luminometric method was developed for the determination of antioxidative activity and was subsequently applied to anthocyanin and betalaine colour concentrates (Kuchta et al., 1999). The method involved quantification of the interruption in luminescence from the hydrogen peroxide-horse radish peroxidase-luminol system in the presence of antioxidants. 

Superoxide radical anion, hydroxyl radical, and hydrogen peroxide are known as prooxidants, whereas substances that neutralize their effects are called antioxidants. Oxidative stress occurs when the prooxidant-antioxidant balance becomes too favorable to the prooxidants. The effects of prooxidants can be neutralized by their direct reaction with small-molecule antioxidants, including glutathione, ascorbate, and tocopherols. In addition, oxidizing radicals are scavenged from a living system by several enzymes, including peroxidase, superoxide dismutase, and catalase. Oxidative lesions on DNA may be repaired by DNA repair enzymes. 

Hiner AN, Rodriguez-Lopez JN, Amao MB et al (2000) Kinetic study of the inactivation of ascorbate peroxidase by hydrogen peroxide. Biochem J 348 321-328... 

Hiner ANP, Martmez JI, Amao MB et al (2001) Detection of a tryptophan radical in the reaction of ascorbate peroxidase with hydrogen peroxide. Eur J Biochem 268 3091-3098... 

Other substrates for the peroxidase will compete with the scopoletin, thus an underestimate of hydrogen peroxide production will result. Thus large amounts of ascorbate will interfere as will thiols. 

Ascorbate peroxidase is a heme-eontaining enzyme that catalyses the ascorbate-dependent reduction of potentially damaging hydrogen peroxide in plants and algae, Eq. (1),... 

Hossain, M. A., and Asada, K., 1984, Inactivation of ascorbate peroxidase in spinach chloroplasts on dark addition of hydrogen peroxide its protection by ascorbate, Plant Cell Physiol. 25 128591295. 

Miyake, C., and Asada, K., 1996, Inactivation mechanism of ascorbate peroxidase at low concentrations of ascorbate hydrogen peroxide decomposes Compound 1 of ascorbate peroxidase. Plant Cell Physiol. 37 423n430. 

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