Fluorometric assay for tissue transglutaminase-mediated transamidation activity.
Herein, we report the development of a direct discontinuous fluorometric transamidation assay for determining tissue transglutaminase (TG2) activity. In the assay reaction, TG2 catalyzes the formation of a biotin-fluorophore conjugate, using a fluorescent, high affinity gamma-glutamyl donor substrate and a biotinylated amine as a gamma-glutamyl acceptor substrate. After the reaction, the conjugate is fixed on streptavidin-coated beads and excess substrate is washed away, allowing the transamidation activity to be quantified by fluorescence measurement. This method was used to detect the activity of as little as 0.6 mU of purified TG2, and can be used for detection of activity from crude cellular lysates. Furthermore, this assay can be used for screening potential inhibitors and synthetic substrates, the latter of which was demonstrated herein.
Gnaccarini
Claudio
C
Département de chimie, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, Canada H3C 3J7.
Ben-Tahar
Wajih
W
Lubell
William D
WD
Pelletier
Joelle N
JN
Keillor
Jeffrey W
JW
D000588:Amines / Q000737:chemistry; D019921:Biotinylation; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
D000588:Amines; D005456:Fluorescent Dyes; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins