Ascorbic acid oxidation of thiol groups from dithiotreitol is mediated by its conversion to dehydroascorbic acid

Area of Science:

• Biochemistry
• Oxidative Stress
• Enzyme Kinetics

Background:

• Ascorbic acid (AA), also known as Vitamin C, is a potent antioxidant but can exhibit pro-oxidant effects under certain conditions.
• The interaction between ascorbic acid and thiol-containing compounds, particularly in biological systems, is complex and not fully understood.
• Understanding the mechanisms of thiol oxidation is crucial for comprehending cellular redox homeostasis and disease pathogenesis.

Purpose of the Study:

• To elucidate whether the in vitro pro-oxidant effect of ascorbic acid on thiol groups is mediated by free radicals or by its oxidized form, dehydroascorbic acid.
• To investigate the role of iron and buffer composition in modulating ascorbic acid-induced thiol oxidation.

Main Methods:

• Measured the oxidation rates of ascorbic acid (AA) and dithiothreitol (DTT), a model for vicinal thiol-containing enzymes, in different buffer systems (MOPS and phosphate buffer).
• Assessed the impact of iron/EDTA complex, superoxide dismutase, catalase, and hydrogen peroxide on AA and DTT oxidation.
• Quantified the effect of iron and EDTA on DTT oxidation in the absence of AA.

Main Results:

• Ascorbic acid and DTT oxidation rates were higher in phosphate buffer compared to MOPS buffer.
• Iron/EDTA-induced AA oxidation was significantly inhibited by superoxide dismutase, catalase, and DTT.
• Iron significantly enhanced AA oxidation, while iron, EDTA, and H2O2 did not promote DTT oxidation independently of AA.

Conclusions:

• In physiological conditions, ascorbic acid's oxidation of thiol groups is mainly driven by its conversion to dehydroascorbic acid, with iron participation.
• Free radicals generated during ascorbic acid auto-oxidation appear to play a minor role in oxidizing thiol groups.
• Buffer composition and the presence of transition metals like iron are critical factors influencing the redox interactions between ascorbic acid and thiols.