ABSTRACT
A fluorescent dipeptide was designed to discover glutamine acceptor proteins of transglutaminase. Starting materials for synthesis were the commercially available compounds carbobenzoxy-L-glutaminylglycine (CBZ-Gln-Gly) and monodansylcadaverine (C-DNS) which were coupled to obtain CBZ-Gln-Gly-C-DNS 1 [1-N-(carbobenzoxy-L-glutaminylglycyl)-5-N- (5'-N', N'-dimethylamino-1'-naphthalenesulfonyl)- diamidopentane]. The glutamine peptide is a substrate of bacterial transglutaminase from Streptoverticillium mobaraense as well as of the guinea pig liver enzyme. This could be shown by incorporating 1 into alpha s1-casein resulting in a significant increase in fluorescence intensity and a concomitant inhibition of cross-linking reaction. Additionally, dipeptide 1 is a useful tool to characterize the specificity of transglutaminase toward small primary amines. We established a sensitive HPLC assay and determined the kinetic parameters of several alkylamines. Hydrolysis of 1 is suppressed in the presence of the nucleophiles as it could be demonstrated with different concentrations of butylamine in semiquantitative studies. Together with labeled primary amines, reagent 1 seems to be a particularly suitable tool for examining acceptor-donor relationships of transglutaminase substrates.
