Abstract
Class III histone deacetylases, or sirtuins, are homologous to the Saccharomyces cerevisiae transcriptional regulator SIR2. The class III enzymes are characterized by their dependence on nicotinamide adenine dinucleotide (NAD+). This cofactor serves as an acetyl-group acceptor in the deacetylation reaction generating O-acetyl-ADP-ribose. Enzymatic activity of sirtuin can be measured in vitro using recombinant proteins purified from mammalian cells after overexpression or after purification from Escherichia coli. This review discusses protocols for the purification of enzymatically active human sirtuin 1, 2, and 3 and their activities on histone and nonhistone substrates.