Fluorometric assay for tissue transglutaminase-mediated transamidation activity

Area of Science:

• Biochemistry
• Enzymology
• Assay Development

Background:

• Tissue transglutaminase (TG2) is a key enzyme involved in various cellular processes.
• Accurate quantification of TG2 activity is crucial for understanding its role in disease and for drug discovery.
• Existing methods for TG2 activity detection may have limitations in sensitivity or applicability to complex samples.

Purpose of the Study:

• To develop a novel, direct, discontinuous fluorometric transamidation assay for quantifying TG2 activity.
• To establish a sensitive and versatile method applicable to purified enzymes and crude cellular lysates.
• To demonstrate the utility of the assay for screening TG2 inhibitors and synthetic substrates.

Main Methods:

• TG2-catalyzed formation of a biotin-fluorophore conjugate using specific donor and acceptor substrates.
• Immobilization of the conjugate on streptavidin-coated beads for separation and washing.
• Quantification of transamidation activity via fluorescence measurement of the bound conjugate.

Main Results:

• The assay demonstrated high sensitivity, detecting as little as 0.6 mU of purified TG2.
• The method proved effective for detecting TG2 activity in crude cellular lysates.
• The assay's applicability for screening potential TG2 inhibitors and novel synthetic substrates was successfully demonstrated.

Conclusions:

• A direct discontinuous fluorometric transamidation assay provides a sensitive and robust method for TG2 activity determination.
• This assay is suitable for both purified enzyme studies and analysis of biological samples.
• The developed assay serves as a valuable tool for biochemical research and high-throughput screening in drug discovery efforts targeting TG2.