Capillary electrophoresis-based sirtuin assay using non-peptide substrates

Sirtuins are NAD+-dependent class III histone deacetylases, which catalyze the deacetylation of acetyl-lysine residues of histones and other protein substrates yielding the deacetylated protein, nicotinamide and 2′-O-acetyl-ADP-ribose. Two lysine amide derivatives containing dansyl (Dns) or 7-dimethylaminocoumarin (DMAC) residues, i.e. Dns-K(Ac)-NH2 and DMAC-K(Ac)-NH2, were synthesized and evaluated as substrates for human sirtuin 1. A CZE method with field amplified sample injection and a MEKC method with sweeping were established and validated for monitoring the deacetylation process of Dns-K(Ac)-NH2 and DMAC-K(Ac)-NH2, respectively. Deacetylation by sirtuin 1 was demonstrated for both of the substrates. The Michaelis-Menten constants, Km, were 88.0 μM for Dns-K(Ac)-NH2 and 42.9 μM for DMAC-K(Ac)-NH2. The applicability of the methods was demonstrated using known sirtuin inhibitors. Resveratrol did not activate sirtuin 1 using the present CE-based enzyme assay. The results indicated that the lysine derivatives can be used in sirtuin assays instead of peptide substrates.