A spectrophotometric assay for histone deacetylase 8 |
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Authors: | Fatkins David G Zheng Weiping |
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Institution: | Department of Chemistry, University of Akron, 190 E. Buchtel Commons, Akron, OH 44325, USA. |
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Abstract: | Inhibitors for the classical protein deacetylase enzymes have been actively pursued to develop the next generation of cancer therapy. Developing a novel convenient assay platform for the classical enzyme-catalyzed reactions could thus facilitate the drug discovery process. Based on our previous studies demonstrating the functional mimicry of N(epsilon)-thioacetyl-lysine for N(epsilon)-acetyl-lysine in the reaction catalyzed by the classical enzyme histone deacetylase 8 (HDAC8) on a peptide template derived from the C terminus of the human p53 tumor suppressor protein, we have developed a spectrophotometric HDAC8 assay via quantifying thioacetate produced from the enzymatic dethioacetylation with Ellman's reagent 5,5'-dithiobis(2-nitrobenzoate). We further demonstrated that this novel assay was selective for HDAC8 versus HDAC1 and 2 and for other classical protein deacetylase enzymes present in the HeLa nuclear extracts, thus making it potentially suitable not only for screening HDAC8-selective inhibitors but also for selectively assessing HDAC8 activity under (patho)physiological conditions. |
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Keywords: | HDAC8 Spectrophotometric assay DTNB |
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