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Related Experiment Videos

A sensitive fluorometric assay for tissue transglutaminase.

T M Jeitner1, H L Fuchsbauer, J P Blass

  • 1Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, New York, 10021, USA. tjeitner@burke.org

Analytical Biochemistry
|May 18, 2001
PubMed
Summary

A new fluorometric assay enables sensitive detection of tissue transglutaminase activity. This high-throughput screening method identifies enzyme inhibitors crucial for drug discovery.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Assay Development

Background:

  • Tissue transglutaminase (TG) plays a role in various biological processes.
  • Developing sensitive assays for TG is important for understanding its function and for drug discovery.
  • High-throughput screening (HTS) requires robust and sensitive assay methods.

Purpose of the Study:

  • To develop a novel, highly sensitive fluorometric assay for tissue transglutaminase.
  • To establish an assay suitable for kinetic analyses and high-throughput screening of enzyme inhibitors.

Main Methods:

  • A fluorometric well plate assay was developed.
  • The assay utilizes a glutaminyl substrate and dansyl cadaverine as an amine substrate.
  • Fluorescence enhancement is measured at specific excitation and emission wavelengths (260 nm and 538 nm, respectively).

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Main Results:

  • The assay demonstrates high sensitivity, capable of detecting as little as 60 microU of purified guinea pig liver tissue transglutaminase.
  • The assay is suitable for multiple kinetic analyses.
  • The assay is amenable to high-throughput screening of chemical libraries for TG inhibitors.

Conclusions:

  • A sensitive and robust fluorometric assay for tissue transglutaminase has been successfully developed.
  • This assay is valuable for kinetic studies and HTS of potential TG inhibitors.
  • The assay's sensitivity and throughput capabilities make it a useful tool in biochemical research and drug discovery efforts.