• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane.

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to remove the acetyl group from a fluorogenic peptide substrate Ac-RGK(Ac)-AMC (Catalog # ) in a coupled assay. The specific activity is >2 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Ser101-Lys399, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  34 kDa

• SDS-PAGE

  30-33 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 25 mM Tris, 150 mM NaCl, 1 mM DTT, pH 9.0

• Stop Buffer: 50 mM Tris, 100 mM NaCl, 30% (v/v) Isopropanol, pH 8.0

• Recombinant Human Sirtuin 3/SIRT3 (rhSIRT3) (Catalog # 7488-DA)

• Fluorogenic Peptide Substrate Ac-Arg-Gly-Lys(Ac)-AMC  (Catalog # ), 20 mM in DMSO

• beta -nicotinamide adenine dinucleotide hydrate ( beta -NAD) (Sigma, Catalog # N6522, 100 mM in diH2O)

• Recombinant Mouse Active Trypsin 3/PRSS3 (Catalog # )

• Nicotinamide (Sigma, Catalog # 72340, 100 mM in diH2O)

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhSIRT3 to 40 ng/μL in Assay Buffer.

2. Dilute Substrate to 1 mM in Assay Buffer containing 2 mM beta -NAD.

3. Load into plate 25 μL of 40 ng/μL rhSIRT3 in triplicate and start the reaction by adding 25 μL of 1 mM Substrate. As a control, load 25 μL of 40 ng/μL rhSIRT3 in triplicate.

4. Seal plate and incubate at 37 °C for 30 minutes.

5. Prepare Stop Solution by combining Recombinant Mouse Trypsin 3/PRSS3 and Nicotinamide in Stop Buffer to final concentrations of 0.2 ng/μL and 4 mM, respectively.

6. Add 50 μL of Stop Solution to all wells. To the control wells, add 25 μL of 1 mM Substrate mixture after addition of Stop Solution.

7. Seal plate tightly with plate sealer and incubate at room temperature for 15 minutes.

8. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode.

9. Calculate specific activity:

     *Adjusted for Control
     **Derived using calibration standard 7-amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A9801).

• rhSIRT3: 1.0 μg

• Substrate: 250 μM

Background: Sirtuin 3/SIRT3

Sirtuin 3 (SIRT3) is a NAD  ⁺-dependent class III histone deacetylase. It is primarily compartmentalized to mitochondria (1) and activates mitochondrial target proteins, including ACSS1, IDH2 and GDH by deacetylating key lysine residues (2-4). SIRT3 plays an important role in regulating mitochondrial metabolism and energy production and thus has emerged as a potential therapeutic target to treat metabolic and neurological diseases (5). Polymorphisms in human SIRT3 have been linked to survivorship among the elderly, suggesting a possible involvement of SIRT3 in age-related phenomena (6).

• References:

1. North, B. J. et al. (2004) Genome Biology 5:224.

2. Schwer, B. et al. (2006) Proc. Natl. Acad. Sci. 103:11224.

3. Schlicker, C. et al. (2008) J. Mol. Biol. 382:790.

4. Ahn, B.-H. et al. (2008) Proc. Natl. Acad. Sci. 105:14447.

5. Jin, L. et al. (2009) J. Biol. Chem. 284: 24394.

6. Rose, G. et al. (2003) Exp. Gerontol. 38: 1065.

• Long Name:

  Silent Mating Type Information Regulation 3 Homolog

• Entrez Gene IDs:

  23410 (Human); 64384 (Mouse); 293615 (Rat)

• Alternate Names:

  EC 3.5.1; EC 3.5.1.-; hSIRT3; mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase; NAD-dependent deacetylase sirtuin-3, mitochondrial; SIR2L3; SIR2L3silent mating type information regulation 2, S.cerevisiae, homolog 3; sir2-like 3; SIR2-like protein 3; SIRT3; sirtuin (silent mating type information regulation 2 homolog) 3 (S. cerevisiae); sirtuin (silent mating type information regulation 2, S.cerevisiae, homolog) 3; Sirtuin 3; sirtuin type 3