A MICROTITER PLATE ASSAY FOR THE CHARACTERIZATION OF SERINE PROTEASESBY THEIR ESTERASE-ACTIVITY

The action of serine (and cysteine) proteases on peptide esters procee ds, as a generalization, orders of magnitude faster than the correspon ding enzymatic hydrolysis of peptide bonds or peptide amides. Esteroly sis liberates an alcohol while generating a free carboxyl group on the peptide; the proton produced can be detected by the use of an appropr iate indicator. The action of trypsin on benzyloxycarbonylalanylargini ne methyl ester was used as a model for the development of a simple mi crotiter plate assay procedure that takes advantage of the speed of th ese reactions and the ease of detection afforded by the color change o f the indicator. A family of ester substrates of the form benzyloxycar bonylalanyl-X-methyl ester, in which X is one of the 20 common amino a cids, was synthesized to allow the determination of the primary specif icity profiles of serine proteases. Using a 96-well microtiter plate t he specificity profiles of four enzymes with all 20 substrates can be carried out in approximately 4 h per enzyme, including setting up and data processing. The primary substrate preferences of trypsin, chymotr ypsin, thrombin, pancreatic elastase, cu-lytic protease, subtilisin, a nd proteinase K were determined to demonstrate the method and were fou nd to be in good general agreement with reported specificities establi shed by more conventional means. (C) 1994 Academic Press, Inc.