A microplate reader-based nonisotopic histone deacetylase activity assay

被引:49
作者
Heltweg, B [1 ]
Jung, M [1 ]
机构
[1] Univ Munster, Dept Pharmaceut Chem, D-48149 Munster, Germany
关键词
histone deacetylase; fluorogenic substrate; BODIPY; 530/550; plate reader;
D O I
10.1006/abio.2001.5542
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 E-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. (C) 2002 Elsevier Science (USA).
引用
收藏
页码:175 / 183
页数:9
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