The design, synthesis and evaluation of active site directed, fluorescent, peptidylchloromethane affinity labels of proteinases



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A series of N- -l-dimethylamino-5-napthalenesulfonyl (dansyl) derivatives of Peptidylchloromethanes (chloromethyl ketones) were synthesized and employed to introduce the fluorescent dansyl moiety specifically into the active sites of proteinases via affinity labelling. Dansylalanyllysylchloromethane (DALCM) was utilized to inactivate and fluorescently label trypsin and the trypsin-like enzymes, thrombin and acrosin. Dansylleucylphenylalanylchloromethane (DLPCM) was synthesized and selectively employed as an inhibitor of chymotrypsin. The di, tri, and tetrapeptides - dansylprolylalanylchloromethane (DPACM) dansylalanylprolylalanylchloromethane (DAPACM), and dansylprolylalanylprolylalanylchloromethane (DPAPACM) - were synthesized and their interaction with elastase evaluated. These fluorescent, fast acting proteinase inhibitors possess enormous potential for the localization, isolation and characterization of enzymes. DALCM, for example, has been used to localize acrosin on the head of spermatozoa. An additional benefit of DALCM lies in its markedly fast rate of trypsin inactivation. This permits the practical utilization of stoichiometric amounts of inhibitor to achieve selective 100% trypsin inactivation even at high dilutions. The transfer of energy between the tryptophan residues of proteinases and a specifically placed dansyl group was used to evaluate proximity relationships in dansylated inhibitor-enzyme ,conjugates of trypsin, chymotrypsin, elastase and thrombin. The anomalies between the mean tryptophan-dansyl distances calculated from fluorescence measurements and the expected mean distances based on x-ray data were explored. The energy transfer studies herein reported provide positive evidence for the conformational homology of a-chymotrypsin and thrombin. A fluorometric enzyme inactivation assay based on energy transfer was developed and employed to determine the rate of inactivation of proteinases by dansylated peptidyl chloromethanes. In this assay the tryptophan residues of the proteinase are excited at 295 nm and the dansyl emission of the developing fluorescent enzyme-inhibitor conjugate is monitored as a function of time.



Chloromethyl ketone, Energy transfer, Fluorometric enzyme inactivation assay, Enzyme localization, Fluorescent affinity label, Trypsin inhibitor