The fluorescence-based acetylation assay using thiol-sensitive probes

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Lysine acetyltransferases (KATs) are crucial enzymes regulating cellular processes through protein acetylation.
• Existing methods for studying KAT activity include radioisotopic labeling, spectrophotometry, and immunoassays.
• Fluorescent methods offer advantages in simplicity, speed, sensitivity, and signal-to-noise ratio for enzymatic assays.

Purpose of the Study:

• To describe technical protocols for a novel fluorescence-based assay to analyze KAT enzymatic activity.
• To demonstrate the utility of thiol-sensitive fluorogenic probes for KAT activity detection.
• To validate the assay using males on the first (MOF) as an exemplary KAT enzyme.

Main Methods:

• Utilized thiol-sensitive fluorogenic probes, specifically 7-Diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM).
• CPM was chosen for its rapid reaction kinetics with coenzyme A (CoA) and high fluorogenicity upon thiol conjugation.
• Developed a fluorescence-based acetylation assay protocol for KAT activity measurement.

Main Results:

• The developed fluorescence assay provides a sensitive and efficient method for analyzing KAT enzymatic activity.
• The assay demonstrated suitability for kinetic characterization of KAT catalysis.
• The method proved effective for investigating KAT inhibitors.

Conclusions:

• Fluorescence-based analysis using thiol-sensitive probes offers a robust platform for studying KAT enzymes.
• This assay facilitates both kinetic studies and the screening of KAT inhibitors.
• The described protocol provides a valuable tool for biochemical and pharmacological research involving KATs.