Expanding on its offerings of long wavelength, highly sensitive SIRT assays, AnaSpec, Eurogentec Group, has released 2 assay kits for SIRT2, the SensoLyte 520 FRET SIRT2 Assay Kit and the SensoLyte Green SIRT2 Assay Kit.
The substrate found in the SensoLyte 520 FRET SIRT2 Assay Kit employs a FRET substrate, and the peptide sequence is derived from a human alpha-tubulin sequence surrounding the deacetylation site of SIRT2. The SensoLyte Green SIRT2 Assay Kit provides a fluorogenic substrate with a human p53 derived sequence.
Compared with lower emitting substrates found in existing commercial kits, these kits employ fluorimetric substrates that emit in the green range of 520 nm. At this higher wavelength, the contribution of autofluorescence is minimal. These kits provide a convenient, two-step homogeneous procedure for measuring sirtuin 2 activity and for screening enzyme inhibitors and activators.
In the first step, an acetylated substrate is incubated with sirtuin-containing samples. In the second step, deacetylated substrate is cleaved by the sirtuin developer, resulting in an increase of fluorescence that can be detected with excitation at 490nm and emission at 520nm. Fluorescence produced is proportional to SIRT2 activity.
Histone deacetylases (HDACs) act as transcriptional repressors of genes catalyzing the removal of acetyl groups from a ε-N-acetyl lysine of histone1. Sirtuins comprise a unique class of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases (class III HDACs) that target multiple protein substrates to execute diverse biological functions. Sirtuins catalyze a reaction that couples lysine deacetylation to NAD hydrolysis, yielding O-acetyl-ADP-ribose and nicotinamide2.
Sirtuin 2 (SIRT2) belongs to the family of Sir2 (Silent Information Regulator 2) proteins. Substrates for SIRT2 are not limited to histones, but also include various transcription factors such as forkhead box (FOXO) transcription factors (FOXO1, FOXO3a) that modulate metabolic, cell cycle and cell death related pathways3, 4. Other prominent substrates include the tumor suppressor p53 and alpha-tubulin5, 6. Human SIRT2 is a cytoplasmic protein that increases in abundance during mitosis and regulates major events of cytokinesis7.
1. Sterner, DE. et al. Microbiol. Mol. Biol. Rev. 64, 435 (2000).
2. Longo, V and Kennedy, B. Cell. 126, 257 (2006).
3. Wang, F. et al. Aging Cell 6, 505 (2007).
4. Jing, E. et al. Cell Metab. 6, 105 (2007).
5. Jin, Y.-H. et al. Biochem. Biophys. Res. Commun. 368, 690 (2008).
6. Inoue, T. et al. Cell Cycle 6(9) (2007) .
7. Dryden, S. et al. Mol. Cell. Biol. 23, 3173 (2003).
AnaSpec, Eurogentec Group