Oxidation of delta-ALA-D and DTT mediated by ascorbic acid: modulation by buffers depends on free iron.
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.
Rocha
João Batista Teixeira
JB
Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. jbtrocha@yahoo.com.br
Lissner
Leandro Ademar
LA
Puntel
Robson Luiz
RL
Fachinetto
Roselei
R
Emanuelli
Tatiana
T
Nogueira
Cristina Wayne
CW
Soares
Félix Alexandre Antunes
FA
D001205:Ascorbic Acid / Q000737:chemistry*; D002021:Buffers; D004229:Dithiothreitol / Q000378:metabolism*; D007501:Iron / Q000737:chemistry*; D010084:Oxidation-Reduction; D000623:Porphobilinogen Synthase / Q000378:metabolism*
D002021:Buffers; D007501:Iron; D000623:Porphobilinogen Synthase; D001205:Ascorbic Acid; D004229:Dithiothreitol