title: High-throughput assays for sirtuin enzymes: a microfluidic mobility shift assay and a bioluminescence assay.
abstract: Silent information regulator or sirtuin (SIRT) enzymes are beta-nicotinamide adenine dinucleotide (oxidized) (NAD(+))-dependent class III histone deacetylases. In this paper, two distinct assays to measure SIRT1 activity are described: a microfluidic mobility shift assay utilizing a fluorophore-labeled peptide substrate and a bioluminescence assay based upon quantitation of remaining NAD(+). The mobility shift assay involves the electrophoretic separation of an N-acetyl-lysine-containing peptide substrate from deacetylated product which bears an additional positive charge. Interference from fluorescent compounds is minimized during screening by direct visualization of separated fluorophore-labeled substrate and product. A preferred peptide substrate for SIRT1 was identified using this assay. The NAD(+) bioluminescence assay couples NAD(+) consumption to the bacterial luciferase-catalyzed oxidation of decanal. This assay does not require synthesis of a labeled peptide and is applicable to sirtuins of any specificity with respect to peptide substrate. The stoichiometry between NAD(+) consumption and peptide deacetylation was shown to be 1:1 by the NAD(+) bioluminescence assay. Kinetic parameters of peptide and NAD(+) cosubstrates and IC(50) values of standard reference inhibitors determined in either assay were similar. With robust Z' values (0.7), both assays are amenable to high-throughput screening.
authors: Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA. Liu.Yichin@gene.com
authors: Yichin
authors: Y
authors: Liu
authors: Raphaele
authors: R
authors: Gerber
authors: John
authors: J
authors: Wu
authors: Trace
authors: T
authors: Tsuruda
authors: John D
authors: JD
authors: McCarter
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D001681:Biological Assay / Q000379:methods*; D002384:Catalysis; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D044085:Microfluidics / Q000379:methods*; D008969:Molecular Sequence Data; D009243:NAD / Q000032:analysis / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D056564:Sirtuin 1; D037761:Sirtuins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D010455:Peptides; D009243:NAD; C447939:SIRT1 protein, human; D056564:Sirtuin 1; D037761:Sirtuins