Fan Yi, Ludewig Ronny, Imhof Diana, Scriba Gerhard K E
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Jena, Philosophenweg 14, Jena, Germany.
Electrophoresis. 2008 Sep;29(18):3717-23. doi: 10.1002/elps.200800361.
Sirtuins are a family of nicotinamide adenine dinucleotide (NAD(+))-dependent enzymes catalyzing the deacetylation of acetyl-lysine residues of histones and other proteins. Three 9-fluorenylmethoxycarbonyl (Fmoc)-labeled peptide substrates derived from the amino acid sequence of p53, i.e. Fmoc-KK(Ac)-NH(2), Fmoc-KK(Ac)L-NH(2) and Fmoc-RHKK(Ac)-NH(2), were synthesized and evaluated as substrates of the human isoenzyme SIRT1. The acetylated and respective deacetylated peptides as well as nicotinamide as the reaction product of nicotinamide adenine dinucleotide were separated by capillary electrophoresis in a fused-silica capillary using 200 mM phosphate-Tris buffer, pH 2.7. Sodium hydroxide-mediated sample stacking was performed in order to overcome peak asymmetry due to the high salt and acid content of the sample as well as to enhance UV detection sensitivity. The assay was subsequently validated. Upon incubation of the acetylated peptides for 60 min in the presence of 2.5 U of SIRT1 at least 87% of the peptides was deacetylated, indicating that the new derivatives are efficient substrates of the enzyme.
沉默调节蛋白是一类依赖烟酰胺腺嘌呤二核苷酸（NAD(+)）的酶，可催化组蛋白和其他蛋白质的乙酰赖氨酸残基的去乙酰化反应。合成了三种源自p53氨基酸序列的9-芴甲氧羰基（Fmoc）标记的肽底物，即Fmoc-KK(Ac)-NH(2)、Fmoc-KK(Ac)L-NH(2)和Fmoc-RHKK(Ac)-NH(2)，并将其作为人同工酶SIRT1的底物进行评估。使用200 mM磷酸盐- Tris缓冲液（pH 2.7），通过熔融石英毛细管中的毛细管电泳分离乙酰化和相应的去乙酰化肽以及作为烟酰胺腺嘌呤二核苷酸反应产物的烟酰胺。进行氢氧化钠介导的样品堆积，以克服由于样品中高盐和酸含量导致的峰不对称，并提高紫外检测灵敏度。随后对该测定法进行了验证。在2.5 U SIRT1存在下，将乙酰化肽温育60分钟后，至少87%的肽被去乙酰化，表明新衍生物是该酶的有效底物。
