Modification of transglutaminase assay: use of ammonium sulfate to stop the reaction

An important modification was made of the assay for transglutaminase regarding dansyl cadaverine incorporation into casein. It is known that the amine, after incorporation into protein by transglutaminase, shows a marked increase of fluorescence accompanied by a slight blue shift. However, measurement of protein-bound fluorescence requires a rather complicated procedure, such as the precipitation by trichloroacetic acid or continually monitoring the fluorescence. To widen the applicability of the method, we utilized an excess concentration of ammonium sulfate to stop the reaction. At concentrations higher than 5 mM, the incorporation of the amine was completely stopped and the fluorescence was retained for more than 2 h. The fluorescence can be measured directly after stopping the reaction, so it has become feasible to assay many samples at a time. Furthermore, the sensitivity and reproducibility of the data were improved, since the reaction time could be prolonged and strictly defined.