Abstract
l-Ascorbate, dehydro-l-ascorbic acid (DHA), and 2,3-diketo-l-gulonate (DKG) can all quench reactive oxygen species (ROS) in plants and animals. The vitamin C oxidation products thereby formed are investigated here. DHA and DKG were incubated aerobically at pH 4.7 with peroxide (H2O2), ‘superoxide’ (a ∼50 : 50 mixture of and ), hydroxyl radicals (•OH, formed in Fenton mixtures), and illuminated riboflavin (generating singlet oxygen, 1O2). Products were monitored electrophoretically. DHA quenched H2O2 far more effectively than superoxide, but the main products in both cases were 4-O-oxalyl-l-threonate (4-OxT) and smaller amounts of 3-OxT and OxA + threonate. H2O2, but not superoxide, also yielded cyclic-OxT. Dilute Fenton mixture almost completely oxidised a 50-fold excess of DHA, indicating that it generated oxidant(s) greatly exceeding the theoretical •OH yield; it yielded oxalate, threonate, and OxT. 1O2 had no effect on DHA. DKG was oxidatively decarboxylated by H2O2, Fenton mixture, and 1O2, forming a newly characterised product, 2-oxo-l-threo-pentonate (OTP; ‘2-keto-l-xylonate’). Superoxide yielded negligible OTP. Prolonged H2O2 treatment oxidatively decarboxylated OTP to threonate. Oxidation of DKG by H2O2, Fenton mixture, or 1O2 also gave traces of 4-OxT but no detectable 3-OxT or cyclic-OxT. In conclusion, DHA and DKG yield different oxidation products when attacked by different ROS. DHA is more readily oxidised by H2O2 and superoxide; DKG more readily by 1O2. The diverse products are potential signals, enabling organisms to respond appropriately to diverse stresses. Also, the reaction-product ‘fingerprints’ are analytically useful, indicating which ROS are acting in vivo.
Highlights
Ascorbate (AA; one form of vitamin C) is the major low-molecular-weight, water-soluble antioxidant in plants and animals, acting to quench reactive oxygen species (ROS)
Treatment of DKG with H2O2 yielded at least five products: CPA [(formerly called compound E) proposed to be 2-carboxy-L-threo-pentonate (i.e. ‘2-carboxy-L-xylonate’ or ‘2-carboxy-L-lyxonate’, which are synonyms)], CPL [(formerly called compound C) proposed to be a mixture of 2-carboxy-L-threo-pentonolactones (2-carboxy-L-xylonolactone plus 2-carboxy-L-lyxonolactone)], an unidentified compound (‘H’ of Parsons & Fry [18]), threonate, and a product that electrophoresed in the oxalyl threonate (OxT) zone (Figure 4; discussed in more detail )
In the present work, we investigated the chemistry of the two DKG oxidation products: H and the putative OxT
Summary
Ascorbate (AA; one form of vitamin C) is the major low-molecular-weight, water-soluble antioxidant in plants and animals, acting to quench reactive oxygen species (ROS). It is present in all metabolically active cell types and sub-cellular compartments, and some AA is released into blood plasma [1] and the plant apoplast (solution which permeates the cell wall) [2,3,4,5,6]. Investigations of the fate of DHA under oxidising conditions in vitro revealed the probable existence of a short-lived, highly reactive intermediate ( proposed to be cyclic-2,3-oxalyl-L-threonolactone; cOxTL), which simultaneously forms three major end-products (Figure 1): cyclic oxalyl threonate (cOxT), oxalyl threonate (OxT), and OxA ( plus ThrO), in a ∼6 : 1 : 1 ratio [17,18]. Transacylase activities can transfer the oxalyl group from OxT to acceptor substrates, e.g. carbohydrates [20]
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