A rapid transglutaminase assay for high-throughput screening applications

Yu Wei Wu, Yu Hui Tsai

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Transglutaminases (TGs) are widely distributed enzymes that catalyze posttranslational modification of proteins by Ca2+-dependent cross-linking reactions. The family members of TGs participate in many significant processes of biological functions such as tissue regeneration, cell differentiation, apoptosis, and certain pathologies. A novel technique for TG activity assay was developed in this study. It was based on the rapid capturing, fluorescence quenching, and fast separation of the unreacted fluorescent molecules from the macromolecular product with magnetic dextran-coated charcoal. As few as 3 ng of guinea pig liver transglutaminase (gpTG) could be detected by the method; activities of 96 TG samples could be measured within an hour. The Km of gpTG determined by this method for monodansylcadaverine (dansyl-CAD) and N,N-dimethylcasein was 14 and 5 μM, respectively. A typical competitive inhibition pattern of cystamine on dansyl-CAD for gpTG activity was also demonstrated. The application of this technique is not limited to the use of dansyl-CAD as the fluorescent substrate of TG; other small fluor-labeled TG substrates may substitute dansyl-CAD. Finally, this method is rapid, highly sensitive, and inexpensive. It is suitable not only for high-throughput screening of enzymes or enzyme inhibitors but also for enzyme kinetic analysis.

Original languageEnglish
Pages (from-to)836-843
Number of pages8
JournalJournal of Biomolecular Screening
Volume11
Issue number7
DOIs
Publication statusPublished - Oct 2006

Keywords

  • Fluorescence
  • Magnetic dextran-coated charcoal
  • TG activity
  • TG kinetics
  • Transglutaminase (TG)

ASJC Scopus subject areas

  • Analytical Chemistry
  • Clinical Biochemistry
  • Biotechnology
  • Biochemistry
  • Molecular Biology

Fingerprint Dive into the research topics of 'A rapid transglutaminase assay for high-throughput screening applications'. Together they form a unique fingerprint.

  • Cite this