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Navigating Side Reactions for Robust Colorimetric Detection of Galactose Oxidase Activity
Ying Sin Koo1, Adrielle Xianwen Chen1, Charlotte Y J Tay1
1Institute of Sustainability for Chemicals, Energy and Environment (ISCE2), Agency for Science, Technology and Research (A*STAR), 8 Biomedical Grove, #07-01 Neuros Building, Singapore 138665, Republic of Singapore.
Analytical Chemistry
|February 28, 2025
Summary
The standard 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay for galactose oxidase is inaccurate due to side reactions. A new 2,4-dinitrophenylhydrazine assay provides accurate enzyme activity screening across a wider range of substrates.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Assay development
Background:
- Colorimetric assays are widely used for enzyme activity screening due to their speed and scalability.
- However, the 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay for galactose oxidase is prone to inaccuracies caused by side reactions and reagent instability.
- These limitations restrict the assay's utility and the range of substrates that can be reliably studied.
Purpose of the Study:
- To identify compounds that interfere with the ABTS colorimetric assay for galactose oxidase activity.
- To elucidate the mechanisms behind these interfering side reactions.
- To develop a more robust and accurate assay for galactose oxidase activity screening.
Main Methods:
- Investigated the incompatibility of specific compound classes (free radical scavengers, phenols, unsaturated aryl ketones) with the ABTS assay.
- Developed a novel assay using 2,4-dinitrophenylhydrazine under neutral conditions with isopropyl alcohol.
- Validated the new assay's compatibility with the previously identified interfering compounds.
Main Results:
- Identified free radical scavengers, phenols with electron-donating substituents, and β,γ-unsaturated aryl ketones as incompatible with the ABTS assay.
- Demonstrated that these compounds cause quenching or adduct formation, leading to inaccurate colorimetric measurements.
- The developed 2,4-dinitrophenylhydrazine assay showed high compatibility with these compounds (R² = 0.98, 0.97, and 0.88, respectively).
Conclusions:
- The ABTS assay for galactose oxidase is unreliable in the presence of certain chemical structures, limiting its application.
- A modified assay using 2,4-dinitrophenylhydrazine under neutral conditions offers a reliable alternative for galactose oxidase activity screening.
- This improved assay expands the substrate scope, facilitating broader applications of galactose oxidase.
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