High-throughput assays for sirtuin enzymes: A microfluidic mobility shift assay and a bioluminescence assay

Silent information regulator or sirtuin (SIRT) enzymes are β-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 IC50 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.