Interference by Mes [2-(4-morpholino)ethanesulfonic acid] and related buffers with phenolic oxidation by peroxidase

Area of Science:

• Biochemistry
• Enzymology

Background:

• Peroxidase enzymes catalyze phenolic oxidation using hydrogen peroxide.
• Apoplastic phenolic compounds are relevant in plant biology and oxidative stress.
• Buffer selection is critical for accurate enzyme kinetic studies.

Purpose of the Study:

• To investigate anomalies observed during kinetic characterization of peroxidase-mediated phenolic oxidation.
• To identify the cause of substrate oxidation underestimation in the presence of Mes buffer.
• To elucidate the mechanism of buffer-induced phenolic substrate recycling.

Main Methods:

• Enzyme kinetics assays using peroxidase and various phenolic substrates.
• Spectrophotometric monitoring of substrate oxidation and hydrogen peroxide utilization.
• Comparative analysis with different buffers (Mes, Hepes, Pipes) and substrate structures.

Main Results:

• Mes buffer (≥10 mM) caused significant recycling of phenolic substrates (<100 µM), leading to apparent underestimation of oxidation.
• Substrate structure influenced recycling; 4'-hydroxyacetophenone showed recycling, while acetosyringone did not.
• The phenoxyl radical intermediate likely interacts with Mes, regenerating the phenol.
• Similar effects were observed with Hepes and Pipes buffers.

Conclusions:

• Mes, Hepes, and Pipes buffers can interfere with peroxidase assays by inducing substrate recycling.
• Careful consideration of buffer effects is essential when interpreting peroxidase kinetics, especially under oxidizing conditions.
• These findings highlight potential pitfalls in biochemical studies employing zwitterionic buffers with radical-generating reactions.