A rapid and sensitive assay method for measuring amine oxidase based on hydrogen peroxide-titanium complex formation

Area of Science:

• Biochemistry
• Plant Physiology

Background:

• Hydrogen peroxide (H₂O₂) is a key product of diamine and polyamine oxidation by oxidase enzymes.
• Accurate measurement of H₂O₂ production is crucial for understanding polyamine metabolism.

Purpose of the Study:

• To develop a novel, sensitive assay for quantifying hydrogen peroxide (H₂O₂) production.
• To optimize assay conditions for measuring diamine oxidase (DAO) and polyamine oxidase (PAO) activity in plant tissues.

Main Methods:

• A new assay based on the formation of a colored H₂O₂-titanium (Ti) complex, measured spectrophotometrically at 410 nm.
• Standardization of assay conditions including pH 7.4, 3 mM substrate, 37°C, 30 min incubation, and catechol as an inhibitor.
• Application to hypocotyl tissues of Vigna catjang and comparison with the starch-iodide method.

Main Results:

• The H₂O₂-Ti complex assay demonstrated significantly higher sensitivity compared to the starch-iodide method.
• Optimized conditions were established for maximum DAO and PAO activity measurement.
• The assay successfully detected DAO/PAO activity in auxin-treated Vigna hypocotyls.

Conclusions:

• The H₂O₂-Ti complex assay provides a more sensitive and reliable method for assessing DAO and PAO activity.
• This assay can be further improved by applying the standardized conditions.
• The method offers a valuable tool for studying polyamine metabolism in plants.