SIRT3 in vitro activity assay

Activity assays were performed as described previously (Hubbard and Sinclair, 2013). Briefly, 100μL reactions containing 0.5 μg purified SIRT3, 100 μM acetylated peptide (APVLFN-K(Ac)-EMIESM, Peptide 2.0), 150 μM NAD+ and 1.3 μg recombinant 6XHis-yPnc1 were incubated at 37° for 1 h in 1 mM DTT/PBS reaction buffer at either pH 6, 7 or 8. Negative control reactions lacking SIRT3 enzyme and positive control reactions containing 1.3 μg recombinant 6XHis-yPnc1 and 150 μM nicotinamide were performed in parallel. 100 μl OPT Developer Solution (10 mM ortho-pthalaldehdye (Sigma Aldrich), 10 mM DTT, 30% ethanol, 70% PBS, pH 7.4) was subsequently added to the reactions and incubated at room temperature for 1 h on an orbital shaker, protected from light. Fluorescence was read on a Varian Cary Eclipse fluorimeter at 420ex/460em wavelengths. Data was normalized to readings for the yPnc1 control reactions at each pH. All reactions were performed in triplicate.