Assays and Substrates

Transglutaminases are defined as protein-glutamine: amine glutamyl transferases (EC 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-carboxamide 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
Transglutaminase reaction pathway

The incorporation of dansylcadaverine into casein (compareable to pathway 2) leads to an increase in fluorescence intensity. The principle is used in kit T036.

During the transpeptidation reaction, ammonia (NH3) is released. The amount of ammonia can be monitored using glutamate dehydrogenase and NADPH as a co-factor. Depending 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. An activity assay using this principle is available for MTG (M001).

The kit M003 is the most sensitive allowing detection of TG2 in the picogram range using the incorporation of biotinylated peptides to microtiter plates displaying primary amines on the surface. 

TG2 - Enzyme Immuno Assay (EIA):
The kit E018 is dedicated to determining the overall quantity of TG2. The principle behind this kit is a sandwich EIA based on two monoclonal antibodies against tissue transglutaminase (TG2). The kit provides coated plates and all the reagents necessary including a tissue transglutaminase calibrator.

The chromogenic assay kit Z009 uses Z-Gln-Gly as the amine acceptor substrate and hydroxylamine as an amine donor. In the presence of transglutaminase, hydroxylamine is incorporated forming glutamyl-hydroxamate, which develops a colored complex with iron (III) detectable at 525 nm (red).

The reverse reaction of TGs is measured in the isopeptidase assay platform including F001 (FXIII), F014 and F016 (TG2).