Development of a scintillation proximity assay for histone deacetylase using a biotinylated peptide derived from histone-H4

Measurement of histone deacetylase activity is usually accomplished by incu bation of the enzyme(s) with acetate-radiolabeled histones or synthetic pep tides based on histone sequences, followed by extraction and quantification of released radiolabeled acetic acid. Consequently, this assay is both tim e consuming and extremely limiting when large numbers of samples are involv ed. We have now developed a simple, two-step histone deacetylase assay that is based on the scintillation proximity assay (SPA) principle. A biotinyla ted [H-3]acetyl histone H4 peptide substrate was synthesized and shown to g enerate a radioactive signal upon binding to streptavidin-coated SPA beads. Incubation of biotinylated [H-3]acetyl peptide with HeLa nuclear extract ( source of histone deacetylase) resulted in a time- and protein-dependent de crease in the SPA signal, providing a measure of enzyme activity. The histo ne deacetylase-mediated decrease in SPA counts was accompanied by a proport ional appearance in free H-3-labeled acetate in the assay mixture. Histone deacetylase activity measured by SPA was concordant with that determined vi a the traditional ethyl acetate extraction procedure. Furthermore, a broad range of histone deacetylase inhibitors was demonstrated to have comparable effects on the catalytic activity of the HeLa nuclei enzyme using both ass ays. The histone deacetylase SPA system described here should be readily ap plicable for automated high-throughput screening and therefore facilitate t he discovery of new inhibitors of histone deacetylases. (C) 1999 Academic P ress.