Rocha João Batista Teixeira, Lissner Leandro Ademar, Puntel Robson Luiz, Fachinetto Roselei, Emanuelli Tatiana, Nogueira Cristina Wayne, Soares Félix Alexandre Antunes
Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil.
Biol Pharm Bull. 2005 Aug;28(8):1485-9. doi: 10.1248/bpb.28.1485.
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.
抗坏血酸（AA）是最重要的内源性还原剂之一，可参与多种细胞活动。在体外，AA在游离金属存在的情况下可作为一种强效氧化剂，在其氧化过程中促进蛋白质结构的修饰并形成活性氧。我们观察到，AA（浓度高于6 mmol/L）会使含巯基的δ-氨基乙酰丙酸脱水酶（δ-ALA-D）失活，并且当在δ-ALA-D活性测定中使用3-吗啉丙磺酸缓冲液（MOPS - pH：6.8；100 mmol/L）而非磷酸钾缓冲液（PB - pH：6.8；100 mmol/L）时，抑制作用会显著降低。δ-ALA-D的抑制作用可能是由金属或肝脏上清液中存在的另一种氧化系统促进的AA自氧化导致的-SH基团氧化介导的。通过研究二硫苏糖醇（DTT - 400 μmol/L）的氧化作为酶巯基的模型，证实了这一假设，我们观察到由抗坏血酸盐引起的DTT和δ-ALA-D氧化的潜在机制是相同的。不同缓冲液之间观察到的差异可能与Fe（II）氧化为Fe（III）有关，这种氧化在PB中比在MOPS缓冲液中更为明显。乙二胺四乙酸（EDTA - 100 μmol/L）和Fe（III）（5 μmol/L）的存在在PB中比在MOPS缓冲液中更能刺激DTT氧化。去铁胺（DF - 100 μmol/L）显著降低了DTT氧化。过氧化氢酶（0.4 mg/ml）和超氧化物歧化酶（SOD - 300 U/ml）对DTT氧化只有适度的影响。目前的结果表明，AA对δ-ALA-D的抑制主要由AA的氧化形式介导，活性氧在该过程中仅起适度作用。
