A fluorometric assay of SIRT1 deacetylation activity through quantification of nicotinamide adenine dinucleotide.
Sirtuins are nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylases that catalyze the deacetylation of proteins such as histones and p53. A sensitive and convenient fluorometric assay for evaluating the SIRT1 enzymatic activity was developed here. Specifically, the remaining NAD(+) after the deacetylation was determined by converting NAD(+) to a highly fluorescent cyclized alpha-adduct compound. By this assay, we found that nicotinamide, Cu(2+), and Zn(2+) antagonize the activity of SIRT1. Resveratrol stimulates the enzymatic activity specifically with 7-amino-4-methylcoumarin (AMC)-labeled acetylated peptide. Epigallocatechin galate (EGCG) inhibits SIRT1 activity with both AMC-labeled and unlabeled peptide. However, a combination of vitamin C with EGCG can reverse the inhibition of EGCG with the unlabeled peptide or stimulate the deacetylation of AMC-labeled peptide by SIRT1. The assay does not require any isotopic material and thus is biologically safe. It can be adapted to a 96-well microplate for high-throughput screening. Notably, the acetylated peptides with or without fluorescent labels may be used in the assay, which facilitates the substrate specificity study of SIRT1 activators or inhibitors in vitro.
Feng
Yu
Y
Department of Molecular Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People's Republic of China.
Wu
Jiahui
J
Chen
Lei
L
Luo
Chen
C
Shen
Xu
X
Chen
Kaixian
K
Jiang
Hualiang
H
Liu
Dongxiang
D
D002392:Catechin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D003374:Coumarins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D009243:NAD / Q000032:analysis*; D010455:Peptides / Q000378:metabolism; D000077185:Resveratrol; D056564:Sirtuin 1 / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013267:Stilbenes / Q000737:chemistry / Q000378:metabolism
D003374:Coumarins; D005456:Fluorescent Dyes; D010455:Peptides; D013267:Stilbenes; D009243:NAD; D002392:Catechin; C045651:epigallocatechin gallate; D056564:Sirtuin 1; C028743:7-amino-4-methylcoumarin; D000077185:Resveratrol