Department of Pharmaceutical Chemistry, School of Pharmacy, University of Jena, Philosophenweg 14, Jena, Germany.
J Pharm Biomed Anal. 2011 Mar 25;54(4):772-8. doi: 10.1016/j.jpba.2010.10.010. Epub 2010 Oct 21.
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.
去乙酰化酶 Sirtuins 是依赖烟酰胺腺嘌呤二核苷酸（NAD(+))的 III 类组蛋白去乙酰化酶，可催化组蛋白和其他蛋白质底物上乙酰化赖氨酸残基的去乙酰化反应，生成去乙酰化蛋白、烟酰胺和 2'-O-乙酰-ADP-核糖。合成了两种含有丹磺酰基（Dns）或 7-二甲基氨基香豆素（DMAC）残基的赖氨酸酰胺衍生物，即 Dns-K(Ac)-NH(2)和 DMAC-K(Ac)-NH(2)，并将其作为人源 Sirtuin 1 的底物进行了评估。建立并验证了毛细管区带电泳（CZE）结合场放大样品进样和胶束电动色谱（MEKC）结合扫集的方法，用于分别监测 Dns-K(Ac)-NH(2)和 DMAC-K(Ac)-NH(2)的去乙酰化过程。两种底物均能被 Sirtuin 1 去乙酰化。Dns-K(Ac)-NH(2 的米氏常数（K(m)）为 88.0μM，DMAC-K(Ac)-NH(2 的 K(m)为 42.9μM。该方法的适用性通过已知的 Sirtuin 抑制剂进行了验证。采用本研究基于 CE 的酶活性测定方法，白藜芦醇不能激活 Sirtuin 1。结果表明，赖氨酸衍生物可用于 Sirtuin 测定，而无需使用肽类底物。
