Real-time fluorescence assay for monitoring transglutaminase activity
Transglutaminases contribute to important processes such as skin cornification or blood coagulation. Therefore, altered transglutaminase activity is linked to diseases. Transglutaminases catalyze the linkage of peptides by transferring the acyl group of proteinogenic glutamine to primary amines like lysine. However, in presence of an excess of accordingly crosslinked substrate, the enzymes catalyze bond hydrolysis.
Here, we show how this is exploited to monitor transglutaminase activity in microplate format. A synthetic substrate carries a fluorophore as well as its quencher, hence not being fluorigenic. If hydrolyzed by the active transglutaminase, quencher and fluorophore are separated leading to an increase in fluorescence that displays transglutaminase activity.
BMG LABTECH's multi-mode plate readers are equipped with reagent injectors to enable CaCl2 to start the reaction and they easily detect changes in fluorescence intensity. The fluorescent model substrate allows for kinetic measurements and identification of transglutaminase regulators.