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A direct fluorometric assay for tissue transglutaminase
Steve M F G Gillet1, Joelle N Pelletier, Jeffrey W Keillor
1Département de Chimie, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, Quebec, Canada H3C 3J7.
Analytical Biochemistry
|November 18, 2005
Summary
A new fluorometric assay was developed to directly measure tissue transglutaminase (TGase) activity. This assay uses a fluorescent substrate and is adaptable for high-throughput screening in microtiter plates.
Area of Science:
- Biochemistry
- Enzymology
- Assay Development
Background:
- Tissue transglutaminase (TGase) plays critical roles in various biological processes.
- Accurate and efficient methods for measuring TGase activity are essential for research and diagnostics.
- Existing TGase assays may have limitations in terms of directness, continuity, or throughput.
Purpose of the Study:
- To design and validate a direct, continuous fluorometric assay for quantifying tissue transglutaminase (TGase) activity.
- To characterize the kinetic parameters of the developed assay using specific TGase substrates.
- To assess the selectivity of the fluorogenic substrate for TGase against other enzymes.
Main Methods:
- Development of a fluorometric assay utilizing 4-(N-carbobenzoxy-l-phenylalanylamino)-butyric acid coumarin-7-yl ester as a substrate.
- Monitoring of 7-hydroxycoumarin release via fluorescence increase (λexc 330 nm, λem 460 nm) to indicate TGase activity.
- Determination of Michaelis-Menten constants (K m) for TGase and its substrates.
- Enzyme selectivity testing against microbial transglutaminase (mTGase), Factor XIIIa, papain, and gamma-glutamyl transpeptidase.
- Adaptation of the assay for a 96-well microtiter plate format.
Main Results:
- A direct and continuous fluorometric assay for TGase activity was successfully established.
- Kinetic parameters (K m) for TGase and its substrates were determined.
- The fluorogenic substrate demonstrated selectivity for TGase over other tested enzymes.
- A modified substrate, 4-(N-carbobenzoxyglycinylamino)-butyric acid coumarin-7-yl ester, also proved effective.
- The assay is compatible with high-throughput 96-well microtiter plate formats.
Conclusions:
- The developed fluorometric assay provides a sensitive, direct, and continuous method for measuring TGase activity.
- The assay's selectivity and adaptability to microtiter plates make it valuable for biochemical research and potential diagnostic applications.
- This method facilitates kinetic studies and screening of TGase modulators.