Specificity assay of serine proteinases by reverse-phase high-performance liquid chromatography analysis of competing oligopeptide substrate library

In this paper we present an HPLC method developed for quick activity and sp ecificity analysis of serine proteinases. The method applies a carefully de signed peptide library in which the individual components differ only at th e potential cleavage site for enzymes. The library has seven members repres enting seven different cleavage sites and it offers substrates for both try psin and chymotrypsin-like enzymes. The individual peptide substrates compe te for the proteinase during the enzymatic reaction. The reaction is monito red by RP-HPLC separation of the components. We describe the systematic des ign of the competitive peptide substrate library and the test of the system with eight different serine proteinases. The specificity profiles of the i nvestigated enzymes as determined by the new method were essentially identi cal to the ones reported in the literature, verifying the ability of the sy stem to characterize substrate specificity. The tests also demonstrated tha t the system could detect even subtle specificity differences of two isofor ms of an enzyme. In addition to recording qualitative specificity profiles, data provided by the system can be analyzed quantitatively, yielding speci ficity constant values. This method can be a useful tool for quick analysis of uncharacterized gene products as well as new forms of enzymes generated by protein engineering. (C) 2001 Academic Press.