A microplate reader-based nonisotopic histone deacetylase activity assay
A microplate reader-based nonisotopic histone deacetylase activity assay
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DOI：
10.1006/abio.2001.5542
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发表时间：
2002-03-15
影响因子：
2.9
通讯作者：
Jung, M
中科院分区：
文献类型：
--
作者：
Heltweg, B;Jung, M
近年来，关于组蛋白去乙酰化酶（HDAC）参与基因调控及其抑制剂在转录治疗中的潜在应用的知识有了极大的增加。这也刺激了对测定HDAC活性和潜在抑制剂效力的新方法的研究。我们之前已经成功地利用e -乙酰赖氨酸的荧光香豆素衍生物开发了一种HDAC的非同位素测定方法。在这里，我们提出基于平板阅读器的定量作为确定底物转换的替代手段。建立了一种新的验证分析程序，使用硼二氮二烯（BODIPY 530/550）而不是香豆素内标，以允许荧光测量而无需色谱分离。该方法的灵敏度、准确度和精密度与已有的高效液相色谱法相当。与一种新的市售的均相板阅读器法相比，HDAC抑制剂trichostatin A的抑制常数相似。市售法具有更高的通量，但其检测HDAC活性的方法不能应用于我们的酶制剂，而我们的底物也被HeLa HDAC转化。这表明我们的系统有更广泛的潜在应用。(C) 2002 Elsevier Science （USA）。
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).