Assays for the measurement of tissue transglutaminase (type II) mediated protein crosslinking via ϵ-( γ-glutamyl) lysine and N′,N′-bis ( γ-glutamyl) polyamine linkages using biotin labelled casein

Abstract

Two colorimetric assays for tissue transglutaminase (type II) activity involving the crosslinking of proteins have been developed. In one assay, biotin labelled casein is crosslinked into chemically modified casein bound to a microtiter plate by tissue transglutaminase and the biotin labelled reaction product is detected by conjugation to Extravidin peroxidase. The assay can detect activity in 10 ng of commercially available purified guinea pig liver transglutaminase and in the crude homogenate derived from 400 human endothelial cells (cell line ECV 304). A correlation ( r 2=0.977) was shown between this assay and the radiolabeled putrescine incorporation assay for the detection of transglutaminase activity. This assay measures the protein crosslinking activity of tissue transglutaminase as opposed to polyamine incorporation and offers a rapid, non-radiometric method for screening large sample numbers. Typical inter-assay variability is 13.9±1.5% ( n=8). In a second assay, the ability of tissue transglutaminase to catalyze the formation of N′,N′-bis ( γ-glutamyl) polyamine bridges is measured. N′,N′-dimethylcasein is bound to a microtiter plate and modified enzymatically using commercially available purified guinea pig liver transglutaminase to incorporate polyamines into glutamine residues. Biotin labelled casein is then crosslinked into the immobilized polyamines by tissue transglutaminase resulting in the formation of N′,N′-bis ( γ-glutamyl) polyamine linkages.