3.3 Assay of SIRT1 Activators Using the PNC1–OPT Assay

The PNC1–OPT assay affords a quick and reliable method to measure the deacetylase activity of SIRT1 without the need for any specialized equipment (Hubbard & Sinclair, 2013). Moreover, in contrast to previously developed SIRT1 assays which require the use of a fluorophore–conjugated peptide substrate, any custom peptide may be used in this assay (Hubbard & Sinclair, 2013). As outlined in Fig. 5, two distinct steps are involved in the measurement of SIRT1 activity using this assay. First, SIRT1 is incubated with acetylated peptide in the presence of β–NAD, reaction buffer, and saturating amounts of PNC1. As nicotinamide is produced from the deacetylation reaction, it is converted into free ammonia (NH₃) by PNC1. Second, the reaction is stopped and the amount of ammonia present is quantified via a chemical reaction with OPT and DTT. This assay can be used to reliably study the effects of any STAC that does not interfere with the fluorescence signal or inhibit PNC1 (Hubbard & Sinclair, 2013).

Outline of the PNC1–OPT assay (Hubbard & Sinclair, 2013). In the first step, deacetylation of a custom peptide substrate by SIRT1 results in the production of nicotinamide (NAM), which is subsequently converted into nicotinic acid and ammonia by the nicotinamidase PNC1. In the second step, the reaction is quenched and ammonia is reacted with o–phthalaldehyde and dithiothreitol (DTT) (in the dark) to produce fluorescent adducts that are quantified using a spectrophotometer.

Thaw an aliquot of nicotinamide and perform a serial dilution to yield solutions that are 10 × of the final reaction concentrations. A series of final concentrations that cover the dynamic range of the assay are 0,5,10, 20, 30, 40, and 50 μM. Pipette 10 μL of each of these solutions into appropriately labeled Eppendorf tubes.

Prepare a reaction mastermix corresponding to the total number of reactions to be performed (it is advisable to perform all reactions in triplicate). For each desired reaction, add 100 μL of assay buffer and 1 μg of PNC1 and mix the solution by gentle vortex.

Pipette 90 μL of the reaction mastermix into each tube containing the nicotinamide standards, mix by pipetting, and close the lid on each tube.

Place all of the sample tubes in a holder rack and shake using an orbital shaker at 37°C in an incubator for 1 h.

During the incubation period, thaw the OPT developer reagent by heating the stock at 42°C. Ensure that the solution is well mixed by vortexing, and that no DTT precipitate is present.

Remove the samples from the incubator, and under dim light, add 100 μL of the OPT developer reagent to each reaction as quickly as possible. Vortex all samples to mix on the highest setting for 5 s. Put the tubes back into a holder, cover all samples with aluminum foil, and incubate at room temperature on an orbital shaker for 1 h (see Note 7).

Transfer samples to a 96–well plate, under dim light, and read the fluorescence using a spectrophotometer with excitation and emission wavelengths set to 413 and 476 nm (Sugawara & Oyama, 1981), respectively (in practice an λ_ex of 420 ± 10 nm and λ_em 460 ± 10 nm work fine a 0.1– 1–s read with or time).

Subtract the background fluorescence (0 μM NAM) from all samples and plot a graph of normalized fluorescence vs concentration of NAM (standard curve).

Thaw aliquots of β–NAD, peptide substrate, and PNC1 and SIRT1 enzymes on ice.

Arrange and label a series of Eppendorf tubes in a holder corresponding to twice the number of samples to be assayed: Label one set “+NAD” and the second set “−NAD.” The latter set of samples will be used as background fluorescence control reactions (see Note 8). In addition, as stated earlier, it is advised that all samples be measured in triplicate (including the −NAD controls).

Pipette the various test compounds into each corresponding tube and include a vehicle control (eg, DMSO).

Prepare a reaction master mix corresponding to the total number of samples to be assayed (both +NAD and −NAD samples). For each reaction add the following (prepare on ice in a 15–mL Falcon tube): Reaction Buffer (100 μL minus the volume of other components), peptide substrate (typically 10–30 μM), PNC1 (1 μg), and SIRT1 purified as described in Section 3.1 (1 μg) (see Note 9). Mix by pipetting followed by gentle vortex (50% efficiency for 5 s).

Divide the mastermix into two Falcon tubes. To one tube add β–NAD to the appropriate final concentration (typically 100 μM) (+β–NAD mastermix), and to the second tube add an identical volume of water (for the no NAD control). Mix briefly.

Aliquot 100 μL of the +NAD mastermix to each experimental reaction tube, and 100 μL of the −NAD mastermix to each corresponding neg ative control reaction. Close the lids on each tube, and very gently vortex to mix (20% amplitude).

Incubate reactions at 37°C for 1 h.

During the incubation period, thaw the OPT developer reagent and incubate at 42°C for ~15 min (keep covered in aluminum foil). Ensure that the solution is well mixed by vortexing, and that no DTT precipitate is observed. If particulates are visible, vortex and continue to heat.

Once the incubation period is complete, under dim light, quickly remove each sample tube from the holder and add 100 μL of OPT developer reagent. If available, a multichannel pipette may be used. It is imperative that the developer be added to each sample as quickly as possible to ensure consistency. Vortex all samples on the highest setting for 5 s.

Place the tubes back into the holder, cover all samples with aluminum foil (to prevent exposure to light), and incubate at room temperature on an orbital shaker for 1 h (see Note 7).

Following the development phase, remove the foil under dim light and transfer 150–200 μL from each tube into 1 well of a 96–well dark bottom plate.

Read the fluorescence using a spectrophotometer with excitation and emission wavelengths set to 413 and 476 nm (Sugawara & Oyama, 1981), respectively (as noted earlier, λ_ex of 420 10 nm and λ_em 460 ± 10 nm work fine with a 0.1– or 1–s read time). Calculate the background fluorescence for each condition by taking the mean of the arbitrary fluorescence (AF) readings for the NAD samples. Next, calculate the net fluorescence for each reaction condition by subtracting the mean background fluorescence from each reading, Fcorrected F_corrected=F_+NAD–F_{−NADcontrol (mean value)}. The resulting value is proportional to the amount of NAM produced during the deacetylation reaction.

AFU can be converted into amounts of NAM production using the linear equation obtained from the standard curve above.