In Vitro Enzymatic Assay and Substrate Specificity

Commonly, in vitro determination of HDAC activity is a manual assay utilizing a coupled two-step process, including enzymatic deacetylation of a substrate followed by reaction termination and readout [10]. Assays utilize nuclear extracts and substrates containing labeled (radioactive or fluorescent) acetylated histones. For the isotope-based assays, the enzymes are incubated with acetate-radiolabled histones prepared from chicken reticulocytes or chemically [ Hjacetylated peptide substrates, and the enzymatic activity is determined by liquid scintillation counting [11]. Alternatively, histones may be obtained from cells following treatment with [ H]acetyl-CoA [12]. The caveats of these approaches include the variability of prelabeled acetylated core histones within preparations, potential high costs, their labor-intensive nature and the presence of radioactive waste. 

Non-isotopic assays have been developed using synthetic acetylated forms of fluorescein-labeled substrates. A number of artificial substrates containing acetylated lysines based on some proteins, such as histones, p53 and p300/CBP, are available for HDAC enzyme activities [11-14]. These include the generic peptides (Fluor-de-Lys), Ac-GA-(NH-Ac-K)-amino-4-methylcoumarin (AMC), Boc-K(Ac)-7-AMC, Ac-NH-GGK(Ac)-AMC [12] and Ac-NH-RHK(Ac)K(Ac)-AMC [14]. The generic peptide 

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