Application of a fluorometric high-performance liquid chromatographic assay for the evaluation of substrates for and inhibitors of transglutaminase

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity is described. The method uses a small synthetic peptide benzyloxycarbonyl- scL-glutaminylgylcine and the fluorescent amine monodansylcadaverine as substrates. Very small amounts of substrates and enzyme are required for this assay. The reaction product is separated from substrates on a reversed-phase, C-18 column, using an isocratic elution solvent consisting of 50% methanol in water, and is detected fluorometrically with didansylcadaverine as standard. An apparent K;Application of this method allowed the evaluation of different compounds as potential transglutaminase inhibitors. A series of alkylaldehydes exhibited inhibition of transglutaminase in a concentration dependent manner, although the results did not parallel the activity of structurally related substrates. An examination of the thioamides, thioacetamide, thioacetanilide, and thiobenzamide showed that all of these compounds inhibited the enzyme activity. The results of time study of thioacetanilide inhibition demonstrated that this compound is an irreversible inhibitor. Two synthetic thioamides, Z- scL-thioGln and Z- scL-thioGlnGly, analogs of transglutaminase substrates were synthesized and evaluated. Both compounds were demonstrated to be transglutaminase inhibitors. In both cases the inhibition was not dependent on calcium ions and was progressive with respect to time, indicating irreversible inhibition. An evaluation of kinetic parameters demonstrated that Z- scL-thioGlnGly is a more effective inhibitor than Z- scL-thioGln. Titration of sulfhydryl groups showed that inactivation of the enzyme with either Z- scL-thioGln or Z- scL-thioGlnGly resulted in the loss of 7-8 sulfhydryl groups per enzyme molecule. Treatment of inactivated enzyme with DTT resulted in its reactivation. The most likely explanation for the chemical basis of inactivation is oxidation of enzyme sulfhydryl groups to disulfides. In the course of these studies, the x-ray crystal structures of Z- scL-thioGln and (2-oxo-6-thioxo-2-piperidinyl)phenylmethyl carbamate (Z- scL-cyclothioGln) were determined.