Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
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Oxidation of δ-ALA-D and DTT Mediated by Ascorbic Acid: Modulation by Buffers Depends on Free Iron
João Batista Teixeira RochaLeandro Ademar LissnerRobson Luiz PuntelRoselei FachinettoTatiana EmanuelliCristina Wayne NogueiraFélix Alexandre Antunes Soares
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2005 Volume 28 Issue 8 Pages 1485-1489

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Abstract

Ascorbic acid (AA) is one of the most important endogenous reducing agents and can participate in a variety of cellular events. In vitro, AA can act as a potent oxidant agent in the presence of free metals, promote modifications in protein structures and form reactive oxygen species during its oxidation. We have observed that AA (above 6 mmol/l) inactivates δ-aminolevulinate dehidratase (δ-ALA-D), a sulfhydryl-containing enzyme and that the inhibitory action was considerably decreased when 3-morpholinepropanesulfonic acid buffer (MOPS – pH: 6.8; 100 mmol/l) was used in the δ-ALA-D activity assay instead of potassium phosphate buffer (PB – pH: 6.8; 100 mmol/l). δ-ALA-D inhibition, probably, is mediated by the oxidation of –SH groups caused by the auto-oxidation of AA promoted by metals or another oxidizing system present in liver supernatants. This hypothesis was confirmed by studying dithiothreitol (DTT – 400 μmol/l) oxidation, as a model of enzyme thiols, where we observed that the mechanism underlying DTT and δ-ALA-D oxidation caused by ascorbate is the same. The difference observed between different buffers may be related to the oxidation of Fe(II) to Fe(III) that was more accentuated in PB than in MOPS buffer. The presence of ethilenediamintetraacetic acid (EDTA – 100 μmol/l) and Fe(III) (5 μmol/l) stimulated DTT oxidation more in PB than in MOPS buffer. Deferroxamine (DF – 100 μmol/l) considerably decreased DTT oxidation. Catalase (0.4 mg/ml) and Superoxide dismutase (SOD – 300 U/ml) had only a modest effect on DTT oxidation. The present results suggest that δ-ALA-D inhibition by AA is mediated primarily by the oxidized form of AA and reactive oxygen species play only a modest role in the process.

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© 2005 The Pharmaceutical Society of Japan
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