A fluorescence anisotropy-based assay for determining the activity of tissue transglutaminase
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Tissue transglutaminase (TGase 2) is the most abundantly expressed enzyme of the transglutaminase family and involved in a large variety of pathological processes, such as neurodegenerative diseases, disorders related to autoimmunity and inflammation as well as tumor growth, progression and metastasis. As a result, TGase 2 represents an attractive target for drug discovery and development, which requires assays that allow for the characterization of modulating agents and are appropriate for high-throughput screening. Herein, we report a fluorescence anisotropy-based approach for the determination of TGase 2’s transamidase activity, following the time-dependent increase in fluorescence anisotropy due to the enzyme-catalyzed incorporation of fluorescein‐ and rhodamine B‐conjugated cadaverines 1–3 (acyl acceptor substrates) into N,N-dimethylated casein (acyl donor substrate). These cadaverine derivatives 1–3 were obtained by solid‐phase synthesis. To allow efficient conjugation of the rhodamine B moiety, different linkers providing secondary amine functions, such as sarcosyl and isonipecotyl, were introduced between the cadaverine and xanthenyl entities in compounds 2 and 3, respectively, with acyl acceptor 3 showing the most optimal substrate properties of the compounds investigated. The assay was validated for the search of both irreversible and reversible TGase 2 inhibitors using the inactivators iodoacetamide and a recently published l‐lysine-derived acrylamide and the allosteric binder GTP, respectively. In addition, the fluorescence anisotropy-based method was proven to be suitable for high-throughput screening (Z′ factor of 0.86) and represents a non-radioactive and highly sensitive assay for determining the active TGase 2 concentration.
KeywordsActive-site titration Cadaverine Enzyme inhibition Fluorescent labeling Transglutaminases Xanthene dyes
Analysis of variance
Blue fluorescent protein
Electrospray ionization mass spectrometry
Theoretical total enzyme concentration
Förster resonance energy transfer
Green fluorescent protein
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
(S)-tert-butyl 6-amino-1-(2-(5-(dimethylamino)naphthalene-1-sulfonamido) ethylamino)-1-oxohexan-2-ylcarbamate (Boc-Lys-en-dansyl)
Nuclear magnetic resonance
Relative fluorescence units
Reversed-phase high-pressure liquid chromatography
Standard error of the mean
25 kDa synaptosome-associated protein
The authors thank Martin Lohse (Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research) for assisting in the synthesis of compound 3. C. H. and M. P. are grateful for support to the Graduate Program in Pharmacology and Experimental Therapeutics of the University of Cologne and the Bayer Health Care AG (Project No. O23). C. H. acknowledges financial support by the Friedrich-Naumann-Stiftung für die Freiheit (ST 6479/P 622). Partial financial support by the Helmholtz Portfolio Topic “Technologie und Medizin—Multimodale Bildgebung zur Aufklärung des in vivo-Verhaltens von polymeren Biomaterialien” (R. W. and R. L.) and by the Fonds der Chemischen Industrie (R. L.) is gratefully acknowledged.
Compliance with ethical standards
Conflict of interest
Funding by the Bayer Health Care AG to C. H. and M. P. has been received via the University of Cologne without any economic obligation. R. W. and R. L. declare that they have no conflict of interest.
Research involving human participants and/or animals and informed consent
This article does not contain any studies with human participants or animals performed by any of the authors. Obtaining informed consent was, therefore, not necessary.
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