Transglutaminases are defined as R-glutaminyl-peptide: amine γ-glutamyltransferase (EC 2.3.2.13). They use a modified double-displacement mechanism to carry out an acyl transfer reaction between the γ-carboxamide group of a peptide-bound glutamine residue and the ε-amino group of a peptide-bound lysine. The active site consists of a catalytic triad (Cys, His and Asp).
The active site cysteine reacts with the γ-carbox-amide of the glutamine, forming a γ-glutamyl thioester releasing ammonia. This activated species subsequently reacts with nucleophilic primary amines, yielding either an isopeptide bond (pathway 1) or a (γ-glutamyl)amine bond (pathway 2). When an amine is not available, the acyl-enzyme intermediate reacts with water to yield glutamic acid (pathway 3).
Reaction pathway of transglutaminase
The incorporation of dansylcadaverine into casein (compare to pathway 2) leads to an increase in fluorescence intensity. The principle is used in kit
T036.
During the transpeptidation reaction ammonia (NH
3) is released. The amount of ammonia can be monitored using glutamate dehydrogenase and NADPH as co-factor. Dependent on the transglutaminase either casein or synthetic peptides serve as acyl donor substrates. The reaction can be monitored online using a UV photometer at 340 nm. Activity assays using this principle for tissue transglutaminase and factor XIII will be available soon.
The most sensitive assay uses the incorporation of biotinylated peptides to microtiter plates displaying primary amines on the surface. The kits
T054 and
T055 include all reagents necessary. After enzymatic incorporation of the biotinylated peptide, biotin is subsequently detected using streptavidin / peroxidase conjugates.
TG2 - Enzyme Immuno Assay (EIA):
The kit
E008 is dedicated to determine the overall TG2-quantity. The principle behind is a sandwich EIA based on two monoclonal antibodies against tissue transglutaminase (TG2). The kit provides coated plates and all reagents necessary including a tissue transglutaminase calibrator.
The chromogenic assay kits
Z009 and
Z010 use different peptides as the amine acceptor substrate and hydroxylamine as amine donor. In the presence of transglutaminase hydoxylamine is incorporated to form glutamyl-hxdroxamate which develops a colored complex with iron (III) detectable at 525 nm (red).
Transglutaminases are able to also cleave the isopeptide bond. This principle is used to provide easy to handle, robust and precise fluorogenic assays suitable for screening of compound libraries and drug development.
