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 incubation of the enzyme(s) with acetate-radiolabeled histones or synthetic peptides based on histone sequences, followed by extraction and quantification of released radiolabeled acetic acid. Consequently, this assay is both time consuming and extremely limiting when large numbers of samples are involved. We have now developed a simple, two-step histone deacetylase assay that is based on the scintillation proximity assay (SPA) principle. A biotinylated [3H]acetyl histone H4 peptide substrate was synthesized and shown to generate a radioactive signal upon binding to streptavidin-coated SPA beads. Incubation of biotinylated [3H]acetyl peptide with HeLa nuclear extract (source of histone deacetylase) resulted in a time- and protein-dependent decrease in the SPA signal, providing a measure of enzyme activity. The histone deacetylase-mediated decrease in SPA counts was accompanied by a proportional appearance in free 3H-labeled acetate in the assay mixture. Histone deacetylase activity measured by SPA was concordant with that determined via 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 assays. The histone deacetylase SPA system described here should be readily applicable for automated high-throughput screening and therefore facilitate the discovery of new inhibitors of histone deacetylases.
Nare
B
B
Department of Parasite Biochemistry and Cell Biology, Merck Research Laboratories, Rahway, New Jersey, 07065-0900, USA. Bakela_Nare@Merck.Com
Allocco
J J
JJ
Kuningas
R
R
Galuska
S
S
Myers
R W
RW
Bednarek
M A
MA
Schmatz
D M
DM
D001710:Biotin; D006367:HeLa Cells; D006655:Histone Deacetylases / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D006801:Humans
D006657:Histones; D001710:Biotin; D006655:Histone Deacetylases