title: Oxidation of delta-ALA-D and DTT mediated by ascorbic acid: modulation by buffers depends on free iron.
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 delta-aminolevulinate dehidratase (delta-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 delta-ALA-D activity assay instead of potassium phosphate buffer (PB - pH: 6.8; 100 mmol/l). delta-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 micromol/l) oxidation, as a model of enzyme thiols, where we observed that the mechanism underlying DTT and delta-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 micromol/l) and Fe(III) (5 micromol/l) stimulated DTT oxidation more in PB than in MOPS buffer. Deferroxamine (DF - 100 micromol/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 delta-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.
authors.affiliation: Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. jbtrocha@yahoo.com.br
authors.forename: João Batista Teixeira
authors.initials: JB
authors.lastname: Rocha
authors.forename: Leandro Ademar
authors.initials: LA
authors.lastname: Lissner
authors.forename: Robson Luiz
authors.initials: RL
authors.lastname: Puntel
authors.forename: Roselei
authors.initials: R
authors.lastname: Fachinetto
authors.forename: Tatiana
authors.initials: T
authors.lastname: Emanuelli
authors.forename: Cristina Wayne
authors.initials: CW
authors.lastname: Nogueira
authors.forename: Félix Alexandre Antunes
authors.initials: FA
authors.lastname: Soares
mesh_terms: D001205:Ascorbic Acid / Q000737:chemistry*; D002021:Buffers; D004229:Dithiothreitol / Q000378:metabolism*; D007501:Iron / Q000737:chemistry*; D010084:Oxidation-Reduction; D000623:Porphobilinogen Synthase / Q000378:metabolism*
chemical_list: D002021:Buffers; D007501:Iron; D000623:Porphobilinogen Synthase; D001205:Ascorbic Acid; D004229:Dithiothreitol