Development of an isoenzyme-specific colorimetric assay for tissue transglutaminase 2 cross-linking activity

作者：Alea Mileidys Perez; Kitamura Miyako; Martin Guillaume; Thomas Vincent; Hitomi Kiyotaka; El Alaoui Said^*

来源：Analytical Biochemistry, 2009, 389(2): 150-156.

DOI：10.1016/j.ab.2009.03.029

• TG2
• Tissue transglutaminase
• Colorimetric assay
• Solid-phase assay
• Spermine
• TG2-specific assay

Tissue transglutaminase (TGase 2) belongs to the multigene transglutaminase family of Ca(2+)-dependent protein cross-linking enzymes. Based on the transamidation activity of TGase 2, a novel colorimetric assay has been developed using covalently coupled spermine to carboxy-substituted polystyrene plates and biotinylated pepT26, an excellent acyl-donor substrate, highly specific for TGase 2. The assay is based on the incorporation of the gamma-carboxamide Of glutamine of pepT26 into the immobilized spermine. The amount of biotinylated pepT26 bound to the plate, as measured by the activity of streptavidin-peroxidase, is directly proportional to the TGase activity. The colorimetric procedure showed a good correlation (r = 0.995) with the commonly used radiometric filter paper method for TGase2, and provides linear dose-response curves over a wide range of hrTGase2 concentrations (2.5-40 mu U/ml). In addition, the assay shows higher sensitivity when compared with our previous TG-colorimetric test (more than 50-fold increase) and other existing assays. PepT26 displays strong reactivity with TGase 2, and no reactivity with TGases 1, 3, and FXIII. The procedure constitutes a rapid, TG2-specific, sensitive, and nonisotopic method for the measurement of TGase 2 activity in as low as 4 ng of hrTGase 2 and purified guinea pig liver transglutaminase, and 1.25 mu g of guinea pig liver extracts.

• 出版日期2009-6-15