Mechanism-based affinity capture of sirtuins

Area of Science:

• Biochemistry
• Chemical Biology
• Enzymology

Background:

• Traditional enzyme activity detection relies on kinetic assays and western blots, often requiring expensive reagents like antibodies.
• Simultaneously detecting enzyme activity and isolating the protein catalyst from mixtures is challenging and often impossible.
• Sirtuins, a family of deacetylases, regulate critical physiological functions such as energy metabolism and DNA repair.

Purpose of the Study:

• To develop a novel chemical approach for the simultaneous detection and isolation of active sirtuins.
• To create new chemical tools for rapid analysis of sirtuin activity in complex biological contexts.
• To establish proof-of-concept tools for studying sirtuins in biological samples.

Main Methods:

• Synthesis of an aminooxy-derivatized NAD(+) and a pan-sirtuin inhibitor.
• Formation of a stable complex between the inhibitor and sirtuin active sites.
• Biotinylation of the sirtuin complex, followed by retrieval using streptavidin beads and gel electrophoresis.

Main Results:

• The chemical tools enable rapid detection and isolation of active sirtuins.
• The method successfully detected and isolated multiple human sirtuin isoforms (SIRT1-3, 5-6) and microbial sirtuins.
• Simultaneous detection of multiple sirtuin isoforms in reaction mixtures was demonstrated.

Conclusions:

• A novel chemical strategy provides a powerful tool for studying sirtuin activity and abundance.
• This method overcomes limitations of traditional techniques for enzyme analysis.
• The developed tools facilitate chemical studies of sirtuins in complex biological samples.