HPLC-Based Enzyme Assays for Sirtuins

  • Jun Young Hong
  • Xiaoyu Zhang
  • Hening LinEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1813)


Sirtuins are a class of enzymes that utilize nicotinamide adenine dinucleotide, NAD+, to remove various acyl groups from protein lysine residues. They have important biological functions and regulate numerous biological pathways. Small molecules that can modulate sirtuin enzymatic activities are potential therapeutic candidates to treat various human diseases. This protocol describes a high-performance liquid chromatography (HPLC)-based method to measure the enzyme kinetics for SIRT2 and SIRT6’s demyristoylase activities and SIRT5’s desuccinylase activity. This method uses peptide substrates that resemble physiological substrates and thus can give more reliable kinetic parameters (Km and kcat values) for these enzymes. The data obtained are useful for understanding the biological function of sirtuins and developing sirtuin modulators.

Key words

Enzymatic activity assay High-performance liquid chromatography assay SIRT2 SIRT5 SIRT6 Demyristoylase Desuccinylase 


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Chemistry and Chemical BiologyCornell UniversityIthacaUSA
  2. 2.Department of Chemistry and Chemical BiologyHoward Hughes Medical Institute, Cornell UniversityIthacaUSA

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