A new fluorescence anisotropy-based assay for activity determination of tissue transglutaminase

Considerable evidence for the implication of tissue transglutaminase (TGase 2) in a variety of pathological processes, such as neurodegenerative diseases, disorders related to autoimmunity and inflammation as well as tumor progression, has been revealed over the recent years. This renders TGase 2 attractive for developing agents which allow the enzyme’s targeting for both therapeutic and imaging purposes. The development of such molecules requires the establishment of reliable methods to assess the interaction with TGase 2, which can be done most conveniently in continuous kinetic assays.
Several assays have been published over the last decades to determine TGase 2 activity, with only very few using the method of fluorescence anisotropy. Measurement of fluorescence anisotropy offers a better signal to noise ratio than other techniques, such as those based solely on fluorescence emission and does not need washing or separation of unbound fluorescent substance.
Here, we report a fluorescence anisotropy-based approach for the determination of TGase 2’s transamidase activity, established and validated by using fluorescein- and rhodamine B-labeled cadaverines as acyl acceptor substrates. The synthesis of the cadaverine derivatives has been accomplished in a solid-phase approach. To allow efficient conjugation of the rhodamine B moiety, different linkers providing secondary amine functions have been introduced between the cadaverine and xanthenyl entities.
The increase in fluorescence anisotropy resulting from covalent binding of the relatively small cadaverine derivatives to the much larger acyl donor substrate N,N-dimethylated casein was followed over time and enzyme activities were derived thereof. The assay was found to be highly reproducible and shows no background signal in the absence of the enzyme for all synthesized cadaverine derivatives. After characterization of the enzyme–substrate interaction by determination of the Michaelis constants, K_m, and the maximum velocities of substrate conversion, V_max, the assay was validated for screening of non-covalent and covalent inhibitors by using the literature-known substances GTP and iodoacetamide, respectively, as well as a recently reported L-lysine acrylamide derivative.