A microplate reader-based nonisotopic histone deacetylase activity assay.
Recent years have brought an enormous increase in knowledge concerning the involvement of histone deacetylase (HDAC) in gene regulation and the potential use of its inhibitors in transcription therapy. This also stimulates research toward new methods for the determination of HDAC activity and thus the potency of potential inhibitors. We have previously succeeded in developing a nonisotopic assay for HDAC using a fluorescent coumarin derivative of epsilon-acetyllysine. Here we present plate reader-based quantitation as an alternative means for the determination of substrate conversion. A new validated assay procedure with a boradiazaindacene (BODIPY 530/550) rather than a coumarin internal standard was established to allow for fluorescence measurement without chromatographic separation. The method is equal in its sensitivity, accuracy, and precision to the previously published HPLC method. A comparison with a new commercially available homogeneous plate reader assay leads to similar inhibition constants for the HDAC inhibitor trichostatin A. The commercial assay has a higher throughput but its procedure for the detection of HDAC activity could not be applied to our enzyme preparation, while our substrate is also converted by HeLa HDAC. This indicates a broader range of potential applications for our system.
Heltweg
Birgit
B
Department of Pharmaceutical Chemistry, Westfälische Wilhelms-Universität Münster, Hittorfstrasse 58-62, 48149, Germany.
Jung
Manfred
M
D000818:Animals; D001896:Boron Compounds / Q000378:metabolism*; D002138:Calibration; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000378:metabolism*; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000032:analysis* / Q000378:metabolism; D006877:Hydroxamic Acids / Q000494:pharmacology*; D051381:Rats; D015203:Reproducibility of Results
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; C012589:trichostatin A; D006655:Histone Deacetylases