Measurement of sirtuin enzyme activity using a substrate-agnostic fluorometric nicotinamide assay

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1077; p. 167
• Main Authors: Hubbard, Basil P, Sinclair, David A
• Format: Journal Article
• Language: English
• Published: United States 01.01.2013
• Subjects: Acetylation; Fluorometry - methods; Humans; Kinetics; Lysine - metabolism; Mitochondrial Proteins - metabolism; NAD - metabolism; Nucleotide Transport Proteins - metabolism; Peptide Fragments - metabolism; Sirtuin 1 - metabolism; Substrate Specificity
• DOI: 10.1007/978-1-62703-637-5_11

The sirtuins are NAD(+)-dependent, multifunctional lysine deacylases that play key roles in cellular homeostasis. They are increasingly being found to target a variety of substrates including acetyl-, butyryl-, malonyl-, and succinyl-lysines. Early assays for measuring sirtuin activity in vitro were criticized for their use of fluorophores on the peptide substrates used, which may alter the results obtained and not be representative of the in vivo situation. We describe a new protocol for the measurement of sirtuin activity by detecting the production of nicotinamide (NAM). The assay is amenable to any substrate and any modification removed by sirtuins. The assay may also be used to measure glycohydrolase (e.g., CD38) and ADP-ribosyltransferase activity (e.g., mARTs and PARPs).