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Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates
14:32

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

Published on: February 27, 2016

Capillary electrophoresis-based sirtuin assay using non-peptide substrates.

Yi Fan1, Martina Hense, Ronny Ludewig

  • 1Department of Pharmaceutical Chemistry, School of Pharmacy, University of Jena, Philosophenweg 14, Jena, Germany.

Journal of Pharmaceutical and Biomedical Analysis
|November 16, 2010
PubMed
Summary

New synthetic substrates, Dns-K(Ac)-NH(2) and DMAC-K(Ac)-NH(2), were developed for studying sirtuins (NAD(+)-dependent histone deacetylases). These novel lysine derivatives enable effective sirtuin 1 activity assays.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Analytical Chemistry

Background:

  • Sirtuins are NAD(+)-dependent class III histone deacetylases involved in various cellular processes.
  • Assaying sirtuin activity typically requires peptide-based substrates, which can be complex to synthesize and use.
  • Developing novel, efficient substrates is crucial for advancing sirtuin research.

Purpose of the Study:

  • To synthesize and characterize novel lysine amide derivatives as potential substrates for sirtuin 1.
  • To establish and validate capillary electrophoresis methods for monitoring sirtuin-mediated deacetylation reactions.
  • To evaluate the utility of these new substrates in sirtuin activity assays and inhibitor screening.

Main Methods:

  • Synthesis of dansyl (Dns) and 7-dimethylaminocoumarin (DMAC) labeled lysine amide derivatives.
  • Capillary Zone Electrophoresis (CZE) with field amplified sample injection for Dns-K(Ac)-NH(2).
  • Micellar Electrokinetic Chromatography (MEKC) with sweeping for DMAC-K(Ac)-NH(2).

Main Results:

  • Successful synthesis and validation of Dns-K(Ac)-NH(2) and DMAC-K(Ac)-NH(2) as sirtuin 1 substrates.
  • Demonstration of sirtuin 1-catalyzed deacetylation for both novel substrates.
  • Determination of Michaelis-Menten constants (K(m)): 88.0μM for Dns-K(Ac)-NH(2) and 42.9μM for DMAC-K(Ac)-NH(2).
  • Resveratrol showed no activation of sirtuin 1 in this assay system.

Conclusions:

  • Lysine amide derivatives are effective substrates for sirtuin 1 activity assays.
  • CE-based methods coupled with these substrates provide a robust platform for sirtuin research.
  • These findings offer an alternative to traditional peptide substrates for sirtuin deacetylation studies.