The determination of histone deacetylase (HDAC) activity and the screening
of potential inhibitors is gaining increasing importance due to the involve
ment of HDAC in transcription regulation. The level of histone acetylation
can be modulated by HDAC inhibitors resulting in differentiation and/or apo
ptosis in cancer cells. We have previously reported the development of a no
nisotopic assay for HDAC using a fluorescent derivative of epsilon -acetyl
lysine. Here we report fluorescein-labeled octapeptides which are substrate
s for HDAC that bear closer resemblance to the native substrate. HPLC with
fluorescence detection is successfully applied to the analysis of the time-
and site-dependent deacetylation. LC-MS analyses are used to confirm the f
indings. The observed selectivity toward one of two possible deacetylation
sites might result from steric hindrance by the label but the methodology p
resented here could be applied to similar larger peptides which might be im
proved tools to characterize HDAC site selectivity in vitro.