Ascorbate oxidase source

Table 5.2 contains data about selected copper enzymes from the references noted. It should be understood that enzymes from different sources—that is, azurin from Alcaligenes denitrificans versus Pseudomonas aeruginosa, fungal versus tree laccase, or arthropodan versus molluscan hemocyanin—will differ from each other to various degrees. Azurins have similar tertiary structures—in contrast to arthropodan and molluscan hemocyanins, whose tertiary and quaternary structures show large deviations. Most copper enzymes contain one type of copper center, but laccase, ascorbate oxidase, and ceruloplasmin contain Type I, Type II, and Type III centers. For a more complete and specific listing of copper enzyme properties, see, for instance, the review article by Solomon et al.4... 

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

There are a number of excellent sources of information on copper proteins notable among them is the three-volume series Copper Proteins and Copper Enzymes (Lontie, 1984). A review of the state of structural knowledge in 1985 (Adman, 1985) included only the small blue copper proteins. A brief review of extended X-ray absorption fine structure (EXAFS) work on some of these proteins appeared in 1987 (Hasnain and Garner, 1987). A number of new structures have been solved by X-ray diffraction, and the structures of azurin and plastocyanin have been extended to higher resolution. The new structures include two additional type I proteins (pseudoazurin and cucumber basic blue protein), the type III copper protein hemocyanin, and the multi-copper blue oxidase ascorbate oxidase. Results are now available on a copper-containing nitrite reductase and galactose oxidase. 

Uchiyama et al. [36] used cucumber juice (source of ascorbate oxidase) for the first time as carrier in a flow injection system for the determination of L-ascorbic acid. In another work, the same researchers used banana pulp and spinach leaf solution as a source of polyphenol oxidase (PPO) in a flow injection system for the determination of polyphenols [37]. However, the first biosensor based on vegetable crude extract (homogenate) was constructed by Signori and Fatibello-Filho [38]. In this study, an amperometric biosensor for the determination of phenols was proposed using a crude extract of yam (Alocasia macrohiza)... 

Figure 10.5 Acrylamide and CsCI quenching effects on the ascorbate oxidase emission properties. Steady-state ( ) and dynamic ( ) quenching of ascorbate oxidase by acrylamide (a) and CsCI (b) upon excitation at 293 nm. Source Di Venere, A., Mei, G., Gilardi, G. et al. (1998). European Journal of Biochemistry, 257, 337-343 with permission from Blackwell Publishing Ltd.

This chapter will concentrate mainly on structural and functional aspects of these enzymes with the major emphasis on ascorbate oxidase and laccase. Significant progress has been achieved in the last 10 years the determination of amino acid sequences of all three enzymes, each from several sources, and the X-ray structure of ascorbate oxidase. The new information forms the basis of a much deeper understanding of the function of the enzymes as will be demonstrated in this chapter. 

Ascorbate oxidase is found in higher plants, but cucumber, Cucumis sativus, and green zucchini squash, Cucurbita pepo medullosa, are the most common sources (29). The immunohistochemical localization of ascorbate oxidase in green zucchini reveals that ascorbate oxidase is distributed ubiquitously over vegetative and reproductive organs in all specimens examined (50). Primary structures of ascorbate oxidase from cucumber, C. sativus (51), pumpkin, Cucurbita sp. Ebisu Nankin (52), and zucchini, C.pepo medullosa (53), have recently been reported. 

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. 

Laccases (p-diphenol O2 oxidoreductase EC 1.10.3.2) catalyze the oxidation of p-diphenols with the concurrent reduction of dioxygen to water. However, the actual substrate specificities of laccases are often quite broad and vary with the source of the enzyme [116,117]. Laccases are members of the blue copper oxidase enzyme family. Members of this family have four cupric (Cu +) ions where each of the known magnetic species (type 1, type 2, and type 3) is associated with a single polypeptide chain. In the blue copper oxidases the Cu + domain is highly conserved and, for some time, the crystallographic structure of ascorbate oxidase, another member of this class of enzymes, has provided a good model for the structure of the laccase active site [124,125]. The crystal structure of the Type-2 Cu depleted laccase from Coprinus cinereus at 2.2. A resolution has also been elucidated [126]. 

Figure 342. Red-edge fluorescence emission of ascort>ate c Jdase. Ma3dinini fluorescence emissioii versus excitation wavelength for Ascorbate oxidase (circles) and for ascorbate oxidase in presence of U M CsCI (squares). Solid laies represent the be linear fits of circles. Source. Di Venere, A Mei. G., Gilardi G., RosaK), N. De Matteis, F. McKay, R, Granon, E. and Faiazzi ro, A. 1998. -. J. Bk)chenL2S7, 337-343. Authraizalion of reprint accordnl from Blackwell Piid ing.

Fig. 4.4. Aciylamide and CsCI quenching ecls on the ascorbate oxidase em ssion properties. Steady-state (open squares) and dynamic (Filed squares) quenching of ascoibale oxidase by aciylamide (A) and CsCI (B) upon excitation at 293 nm. Source Di Venerc, A, Mei, C.. Gibrdi, G.. Ros, N., Oe MatteE, F., McKs. R.. Cralton. E. and Finazzi Agro, A. I99S. Fur J. Bioclicm 257. 337-343. AudxHization of reprint accorded by Blackwell PuUisliing.

The blue multicopper oxidases constitute a heterogeneous family of enzymes from different sources (7). In addition to the well characterized members of this family, ascorbate oxidase (45,46), laccase (47,48), and ceruloplasmin (49,50), all from higher organisms, two other proteins have attracted much recent interest FetSp, which is involved in iron uptake in yeast (51), and CueO, which is required for copper homeostasis in Escherichia coli (52). The characteristic reactivity of these enzymes is the one-electron oxidation of four substrate equivalents coupled to the four-electron reduction of dioxygen to water (1). These processes occur at a catalytic unit constituted by four copper atoms classified according to their spectroscopic properties in... 

Clay-modified electrodes with ruthenium purple Enzymic treatment with Cucumis sativus tissue which is a rich source of ascorbate oxidase Based on the quantitative electrolysis of AA A four-channel multipotentiostat for simultaneous measurements with microelectrode arrays. To reduce the complexity of electrochemistry cell, only one reference, and one auxiliary electrode are used Electrocatalytic determination of AA using tetraaza macrocycle-modified electrodes Neutralization of AA in ammonia leading to a change in conductivity... 

Diketogulonate cannot be reduced back to ascorbate. Most animals synthesize ascorbate using the path shown in Fig. 7.7, but primates including humans have accumulated mutations in the gene for L-gulonolactone oxidase and cannot synthesize ascorbate de novo. They require a dietary source of vitamin C to compensate for the gradual loss of dehy-droascorbate. The current Recommended Dietary Allowance (RDA) from the US Food and Drug Administration is 60-95 mg/day. 

Uchida et al. excluded the possible function of ascorbic acid as a peroxide source and considered their enzyme to be an oxidase, not a peroxidase. Their second enzyme converted dihydroxyphenylpyruvic acid to homo-... 

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. 

In contrast to ascorbic acid and a-tocopherol amidothionophosphates did not have any prooxidative effects as measured by oxygen consumption from buffer solutions containing the drug and cupric sulphate as a source of redox-active metal ions (Ti-ROSH et al. 1996). Amidothionophosphates reduced significantly and in a dose-dependent manner the oxygen burst in human neutrophils as measured by luminol-dependent luminescence, and they also markedly depressed the killing of human fibroblasts by mixtures of glucose oxidase and streptolysin S. The toxicity of these molecules was tested by intraperitoneal injection of doses up to 1000 mg/kg to white Sabra mice. No mortality was observed 30 d after administration of up to 500 mg/kg. 

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. 

---