Thin layer chromatography assay to detect laccase inhibitors

Area of Science:

• Biochemistry
• Analytical Chemistry
• Natural Product Discovery

Background:

• Thin-layer chromatography (TLC) hyphenated with bioassays is crucial for identifying bioactive compounds in complex mixtures.
• Laccase inhibitors are important targets in various biological and industrial applications.
• Existing methods for detecting laccase inhibitors on TLC plates are limited.

Purpose of the Study:

• To develop and optimize the first bioautographic assay for detecting laccase inhibitors directly on a TLC plate.
• To differentiate between true laccase inhibitors and radical scavengers using a combined enzymatic and chemical radical generation approach.
• To apply the developed methodology for the detection of hydrazone-based laccase inhibitors.

Main Methods:

• Optimization of the on-plate reaction between laccase and ABTS to generate the detectable ABTS radical cation (ABTS·+).
• Development of a control TLC assay using chemically generated ABTS·+ to distinguish inhibitor types.
• Application of the optimized TLC-bioassay to analyze a series of synthesized hydrazone compounds.

Main Results:

• The optimized TLC-bioassay successfully detected laccase inhibitors with limits of detection and quantification for sodium azide at 54.9 ng and 166 ng, respectively.
• The method effectively differentiated between the specific laccase inhibitor sodium azide and radical scavengers like gallic and kojic acids.
• The assay was successfully applied to identify and detect individual laccase inhibitors within the hydrazone chemotype.

Conclusions:

• A novel and effective bioautographic TLC assay for the specific detection of laccase inhibitors has been established.
• This method provides a powerful tool for the characterization of laccase inhibitors and the discovery of new bioactive compounds.
• The developed technique enables the individual detection of laccase inhibitors separated on TLC, advancing natural product research.