title: Capillary electrophoresis-based sirtuin assay using non-peptide substrates.
abstract: 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)-NH(2) and DMAC-K(Ac)-NH(2), 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)-NH(2) and DMAC-K(Ac)-NH(2), respectively. Deacetylation by sirtuin 1 was demonstrated for both of the substrates. The Michaelis-Menten constants, K(m), were 88.0μM for Dns-K(Ac)-NH(2) and 42.9μM for DMAC-K(Ac)-NH(2). 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.
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of Jena, Philosophenweg 14, Jena, Germany.
authors.forename: Yi
authors.initials: Y
authors.lastname: Fan
authors.forename: Martina
authors.initials: M
authors.lastname: Hense
authors.forename: Ronny
authors.initials: R
authors.lastname: Ludewig
authors.forename: Christin
authors.initials: C
authors.lastname: Weisgerber
authors.forename: Gerhard K E
authors.initials: GK
authors.lastname: Scriba
mesh_terms: D055162:Biocatalysis; D020374:Chromatography, Micellar Electrokinetic Capillary; D003374:Coumarins / Q000737:chemistry; D003619:Dansyl Compounds / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods; D019075:Electrophoresis, Capillary; D004789:Enzyme Activation / Q000187:drug effects; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D015203:Reproducibility of Results; D056564:Sirtuin 1 / Q000378:metabolism; D037761:Sirtuins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity; D013678:Technology, Pharmaceutical
chemical_list: D003374:Coumarins; D003619:Dansyl Compounds; D056572:Histone Deacetylase Inhibitors; D007202:Indicators and Reagents; D011994:Recombinant Proteins; C092582:7-dimethylamino-4-methylcoumarin; C447939:SIRT1 protein, human; D056564:Sirtuin 1; D037761:Sirtuins; D008239:Lysine