Application of a fluorescence anisotropy-based assay to quantify transglutaminase 2 activity in cell lysates

Transglutaminase 2 (TGase 2) represents a multifunctional protein, which is involved in various physiological and pathophysiological processes. The latter also include its participation in the development and progression of malignant neoplasms, which is often accompanied by an in-creased protein synthesis. Besides the elucidation of the molecular functions of TGase 2 in tumor cells, knowledge of its concentration that is available for targeting by theranostic agents repre-sents a valuable information. Herein, we describe the application of a recently developed fluo-rescence anisotropy (FA)-based assay for the quantitative expression profiling of TGase 2 by means of transamidase-active enzyme in cell lysates. The assay is based on the incorporation of rhodamine B‑isonipecotyl‑cadaverine (R-I-Cad) into N,N-dimethylated casein (DMC), which results in an increase of FA signal over time. It was shown that this reaction is not only catalyzed by TGase 2 but also by TGases 1, 3, and 6 and factor XIIIa using recombinant proteins. Therefore, control measurements in the presence of a selective irreversible TGase 2 inhibitor were manda-tory to ascertain the specific contribution of TGase 2 to the overall FA rate. To validate the assay regarding the quality of quantification, spike/recovery and linearity of dilution experiments were performed. A total of 25 cancer and 5 non-cancer cell lines were characterized with this as-say method in terms of their activatable TGase 2 concentration (fmol/µg protein lysate) and the results were compared to protein synthesis data obtained by western blotting. Moreover, com-plementary protein quantification methods using a biotinylated irreversible TGase 2 inhibitor as activity-based probe and a commercially available ELISA were applied for selected cell lines to further validate the results obtained by the FA-based assay. Overall, the present study demonstrates that the FA-based assay using the substrate pair R-I-Cad and DMC represents a facile