Ascorbic acid oxidase nature

An ever increasing number of copper metalloproteins is being recognized. Those regularly present in mammals are listed in Table 5 with some of their characteristics. Other important naturally occurring copper proteins, such as hemocyanin, laccase, and ascorbic acid oxidase, are not listed since they do not occur in mammals. The metalloprotein nature of some of the proteins listed in Table 5 has not been established fully as yet. The search for further copper proteins, copper-protein complexes, and other forms in which copper may be stored or transported in the body must continue. 

Certain microorganisms, such as Escherichia coli and Staphylococcus albus strains will reduce dehydroascorbic acid. This reduction has been used for the estimation of ascorbic acid after its oxidation in extracts by ascorbic acid oxidase (S27) and also in the chemical reduction methods as a way of reducing dehydroascorbic acid to ascorbic acid. The reduction is not specific for L-ascorbic acid, but interfering compounds are unlikely in natural products. The use of this reducing action of bacteria has been improved by Mapson and Ingram (M8). 

For those, undoubtedly the majority even in Western countries, who rely on natural dietary sources of the vitamin, care is needed in culinary practice if much of the ascorbic acid is not to be lost. As seen in Chapter 5 the fine cutting of vegetables releases ascorbic acid oxidase which will destroy the vitamin and the use of excessive water for cooking will leach it out of the food. Overcooking and the addition of sodium bicarbonate, thankfully by now little practised, also destroy the vitamin by oxidation which is particularly rapid in cooking pots made of copper. For the human infant, as for any mammal, mother s milk is a whole food and that includes vitamin C at a level of 3.0-5.5 mg%. Bovine milk is much less rich and needs supplementation for feeding to human infants. 

These observations confirm the view that the copper-protein bond in the enzyme is of a nondissociable nature and are not in agreement with the work of Lampitt and Clayson (1945), who postulated that the catalytic activity of ascorbic acid oxidase was to be attributed to traces of ionic copper resulting from an ionization of the copper bound to nonspecific protein. 

Oxidations now known to be catalyzed by copper-containing enzymes were noticed over a century ago, when Schoenbein observed that oxidation of natural substrates resulted in pigment formation in mushrooms. Individual enzymes were gradually identified laccase by Yoshida in 1883 and tyrosinase by Bertrand in 1896. However, it was not imtil potato polyphenol oxidase was isolated in 1937 by Kubowitz that the role of copper was defined. The family of copper oxidases includes a number of enzymes of both plant and animal origin that may very probably be found to react through similar mechanisms, but which exhibit a number of individual characteristics. The enzymes to be described in this section include potato phenol oxidase, mushroom polyphenol oxidase (tyrosinase), laccase, mammalian and insect tyrosinase, and ascorbic acid oxidase. Each of these differs in certain respects from the others, and undoubtedly other related enzymes will be described from other sources that resemble these, but also display individualities. In these cases, identities in nomenclature must not be extended to imply identities in enzyme structure or activity. 

L-ascorbic acid Potentiometric Based on ascorbate oxidase of natural source immobilised on ethylene-vinylacetate membrane Fernandes et al. (1999)... 

Fernandes, J.C.B. Kubota, L.T. Neto, G.d.O. Potentiometric biosensor for L-ascorbic acid based on ascorbate oxidase of natural source immobilized on ethylene-vinylacetate membrane. Anal. Chim. Acta 1999, 385, 3-12. 

Ascorbic acid browning is also inhibited by the addition of sulfite (Wedzicha and McWeeny, 1974). The same holds for polyphenol oxidase-catalyzed oxidation of natural phenols in fruit. The mechanism of the inhibition is by reaction of oquinone intermediates with sulfite, which leads to nonreactive sulfocatechols (Wedzicha, 1995). 

Blue (Type 1) copper proteins are found widely in nature. Typical examples are plasiocyaiiin (MW 10,500) and ascorbale oxidase (MW 150,000) which contain one and eight Cu atoms per protein, respectively. The former serves as a component of the electron transfer chain in plant photosynthesis while the latter is an enzyme involved in the oxidation of ascorbic acid. The oxidized form is characterized by intense blue color due to electronic absorption near 600 nm. In addition, blue copper proteins exhibit unusual properties such as extremely small hyperfine splitting constants (0.003 0.009 cm" ) in ESR spectra and rather high redox potential (4-0.2 0.8 V) compared to the Cu(ll)/Cu(I) couple in aqueous solution. 

There are few references in the literature to the inhibition of oxidase enzymes by naturally occurring substances. The presence of a substance in many fruits and vegetables which effectively inhibits the oxidation of ascorbic acid, whether this is brought about by ascorbic oxidase, poly-phenolase, peroxidase, or by inorganic Cu, has been reported by Somogyi (1944), but the nature of this substance or substances was not identified. Damodaran and Nair (1936) isolated a tannin from the Indian gooseberry (Phyllanthus emblica) which inhibited the oxidation of ascorbic acid in the press juice. Since the protective effect of this substance could be overridden by the addition of Cu, they concluded that its action depended on the suppression of metal catalysis. 

Intermediate formation of qulnone derivatives and cytochrome oxidase via cytochrome C CBonner, 1957 Aberg, 1958 Mapson, 1958 Chlnoy, 1962 Chlnoy et al., 1969 1969 a 1970). In some cases these enzyme systems may readily oxidize all AA to DHA upon grinding while In other cases natural Inhibitors, e g, oxalic acid, strongly retard this process CAberg, 1958 Mapson, 1958). Hence a method for the determination of ascorbic acid utilization (AAU was developed ... 

D-glucose and the three-enzyme system GOx, mutarotase and invertase for sucrose estimation. A common format was adopted to facihtate design and operation, in this case immobilization method, the fact that all enzymes used were oxidases and that a common detection principle, reoxidation of H2O2 generated product, was chosen (except for ascorbic acid which was estimated directly). Pectin, a natural polysaccharide present in plant cells, was used as a novel matrix to enhance enzyme entrapment and stabilization in the sensors. Interferences related to electrochemi-caUy active compounds present in fruits under study were significantly reduced by inclusion of a suitable cellulose acetate membrane diffusional barrier or by enzymatic inactivation with ascorbate oxidase. Enzyme sensors demonstrated expected response with respect to their substrates, on analyte average concentration of 5 mM. 

This progress is mainly due to the determination of the amino-acid sequences for all members of this group and the X-ray crystal structure of ascorbate oxidase. The three-dimensional structure of ascorbate oxidase showed the nature and spatial arrangement of the copper centers and the three-domain structure. However, modern spectroscopic techniques (e.g., low-temperature MCD and ENDOR) made invaluable contributions as well. 

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