A microplate reader-based nonisotopic histone deacetylase activity assay.

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Histone deacetylase (HDAC) enzymes play a crucial role in gene regulation.
• HDAC inhibitors are being investigated for their therapeutic potential in transcription therapy.
• Accurate methods for determining HDAC activity are essential for evaluating inhibitor potency.

Purpose of the Study:

• To develop a novel, non-isotopic, plate reader-based assay for quantifying histone deacetylase (HDAC) activity.
• To establish a validated assay procedure using a BODIPY 530/550 internal standard for fluorescence measurement.
• To compare the performance of the new assay with existing methods, including HPLC and a commercial plate reader assay.

Main Methods:

• Development of a new assay utilizing a fluorescent substrate and a BODIPY 530/550 internal standard.
• Plate reader-based quantitation of substrate conversion without the need for chromatographic separation.
• Validation of the assay for sensitivity, accuracy, and precision, and comparison with HPLC and a commercial assay using trichostatin A as an HDAC inhibitor.

Main Results:

• A new validated assay procedure was established, enabling fluorescence measurement without chromatographic separation.
• The developed method demonstrated comparable sensitivity, accuracy, and precision to the previously published HPLC method.
• Similar inhibition constants for trichostatin A were obtained when compared to a commercial homogeneous plate reader assay, indicating broader applicability.

Conclusions:

• The new plate reader assay provides a sensitive, accurate, and precise alternative for determining HDAC activity and evaluating inhibitor potency.
• This method allows for fluorescence measurement without chromatographic separation, simplifying the assay procedure.
• The assay shows potential for broader applications in HDAC research compared to some commercial alternatives.