Histone deacetylases regulate the acetylation levels of numerous proteins and 
play key roles in physiological processes and disease states. In addition to 
acetyl groups, deacetylases can remove other acyl modifications on lysines, the 
roles and regulation of which are far less understood. A peptide-based 
fluorescent probe for single-reagent, real-time detection of deacetylase 
activity that can be readily adapted for probing broader lysine deacylation, 
including decrotonylation, is reported. Following cleavage of the lysine 
modification, the probe undergoes rapid intramolecular imine formation that 
results in marked optical changes, thus enabling convenient detection of 
deacylase activity with good statistical Z' factors for both absorption and 
fluorescence modalities. The peptide-based design offers broader isozyme scope 
than that of small-molecule analogues, and is suitable for probing both metal- 
and nicotinamide adenine dinucleotide (NAD+ )-dependent deacetylases. With an 
effective sirtuin activity assay in hand, it is demonstrated that iron chelation 
by Sirtinol, a commonly employed sirtuin inhibitor, results in an enhancement in 
the inhibitory activity of the compound that may affect its performance in vivo.