A direct fluorometric assay for tissue transglutaminase

Abstract

Herein we report the design of a direct and continuous fluorometric assay for determining tissue transglutaminase (TGase) activity. The progress of the TGase-catalyzed reaction of 4-( N-carbobenzoxy- l-phenylalanylamino)-butyric acid coumarin-7-yl ester was monitored as an increase of fluorescence ( λ exc 330 nm, λ em 460 nm) due to the release of 7-hydroxycoumarin. Using this assay, we determined the K m of two acceptor substrates, N-acetyl- l-lysine methyl ester and aminoacetonitrile. We also determined the K m of 4-( N-carbobenzoxy- l-phenylalanylamino)-butyric acid coumarin-7-yl ester for its TGase-mediated hydrolysis and for its enzymatic reaction with the acyl acceptor substrates N-acetyl- l-lysine methyl ester and aminoacetonitrile. We ascertained that the fluorescent substrate was selective toward tissue TGase by testing it with different enzymes, namely microbial transglutaminase (mTGase), Factor XIIIa, papain, and γ-glutamyl transpeptidase. 4-( N-carbobenzoxyglycinylamino)-butyric acid coumarin-7-yl ester, lacking the benzyl side chain, was also found to be an efficient fluorogenic substrate of tissue TGase. Finally, we have shown that this method is applicable to 96-well microtiter plate format.