Microplate filtration assay for nicotinamide release from NAD using a boronic acid resin.
We describe a microplate-based assay for NAD-dependent Class III histone deacetylases (also known as SIRTs) that measures the enzyme-catalyzed release of nicotinamide from radiolabeled NAD, using a boronic acid resin to selectively capture the NAD. This method avoids the need for fluorogenic or radiolabeled peptides or separation of the reaction products using solvent extraction. The protocol reported here is rapid and uses commercially available materials. The use of a simple microplate filtration device allows for the simultaneous processing of 96 samples, facilitating enzyme kinetic analyses and inhibition studies. Furthermore, monitoring nicotinamide release rather than peptide deacetylation obviates the need for chemical modification of protein and peptide substrates. This assay is applicable to SIRTs and other enzymes that cleave nicotinamide from NAD.
McDonagh
Thomas
T
Elixir Pharmaceuticals, One Kendall Square, Cambridge, MA 02139, USA.
Hixon
Jeffrey
J
DiStefano
Peter S
PS
Curtis
Rory
R
Napper
Andrew D
AD
D000180:Acrylic Resins / Q000737:chemistry*; D000818:Animals; D001897:Boronic Acids / Q000737:chemistry*; D002250:Carbon Radioisotopes / Q000737:chemistry; D002384:Catalysis; D019411:Clinical Laboratory Techniques; D005374:Filtration / Q000379:methods; D006655:Histone Deacetylases / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D009536:Niacinamide / Q000737:chemistry / Q000378:metabolism*; D037761:Sirtuins / Q000737:chemistry / Q000378:metabolism*
D000180:Acrylic Resins; D001897:Boronic Acids; D002250:Carbon Radioisotopes; D009243:NAD; D009536:Niacinamide; D037761:Sirtuins; D006655:Histone Deacetylases