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A rapid transglutaminase assay for high-throughput screening applications
1Graduate Institute of Pharmacy, Taipei Medical University, Taipei, Taiwan.
Journal of Biomolecular Screening
|August 25, 2006
Summary
A new assay rapidly measures transglutaminase (TG) activity using magnetic charcoal to separate fluorescent molecules. This sensitive, inexpensive method is ideal for high-throughput screening and enzyme kinetics.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Transglutaminases (TGs) are enzymes catalyzing Ca(2+)-dependent protein cross-linking.
- TGs are crucial in biological processes like cell differentiation, apoptosis, and tissue regeneration.
- Dysfunctional TGs are implicated in various pathologies.
Purpose of the Study:
- To develop a novel, rapid, and sensitive assay for transglutaminase (TG) activity.
- To enable high-throughput screening of TG activity and enzyme inhibitors.
- To facilitate enzyme kinetic analysis of TGs.
Main Methods:
- A new TG activity assay utilizing rapid capture, fluorescence quenching, and magnetic separation.
- Magnetic dextran-coated charcoal is used for fast separation of unreacted fluorescent molecules.
- The assay was validated using guinea pig liver transglutaminase (gpTG) and monodansylcadaverine (dansyl-CAD).
Main Results:
- The assay detected as little as 3 ng of gpTG.
- Up to 96 TG samples could be assayed within one hour.
- Determined K(m) values for gpTG were 14 muM (dansyl-CAD) and 5 muM (N,N-dimethylcasein).
- Demonstrated competitive inhibition by cystamine.
Conclusions:
- The developed TG activity assay is rapid, highly sensitive, and cost-effective.
- This method is suitable for high-throughput screening of enzymes and inhibitors.
- The assay is applicable for detailed enzyme kinetic studies of transglutaminases.
