Development of a capillary electrophoresis-based assay of sirtuin enzymes

Sirtuins are a family of nicotinamide adenine dinucleotide (NAD+)-dependent enzymes catalyzing the deacetylation of acetyl-lysine residues of histones and other proteins. Three 9-fluorenylmethoxycarbonyl (Fmoc)-labeled peptide substrates derived from the amino acid sequence of p53, i.e. Fmoc-KK(Ac)-NH2, Fmoc-KK(Ac)L-NH2 and Fmoc-RHKK(Ac)-NH2, were synthesized and evaluated as substrates of the human isoenzyme SIRT1. The acetylated and respective deacetylated peptides as well as nicotinamide as the reaction product of nicotinamide adenine dinucleotide were separated by capillary electrophoresis in a fused-silica capillary using 200 mM phosphate-Tris buffer, pH 2.7. Sodium hydroxide-mediated sample stacking was performed in order to overcome peak asymmetry due to the high salt and acid content of the sample as well as to enhance UV detection sensitivity. The assay was subsequently validated. Upon incubation of the acetylated peptides for 60 min in the presence of 2.5 U of SIRT1 at least 87% of the peptides was deacetylated, indicating that the new derivatives are efficient substrates of the enzyme.