Methodological considerations on chromogenic peptide substrate assays and application on automated analyzers

Chromogenic peptide substrates (CPS) are small peptides with the chromophore p-nitroaniline (pNA) coupled to the terminal carboxyl group in an amide linkage. These substrates are the basis for a new generation of analyses of serine proteases. A short review of established manual methods for determination of key components of the plasma proteolytic enzyme systems as well as other serine proteases is given here. The enzymatic cleavage of the peptide-pNA amide linkage in CPS results in release of pNA. This reaction can be monitored at 405 nm in a spectrophotometer and can easily be automated using equipment found in most clinical chemistry laboratories. In our laboratory we have adapted such assays to the LKB 8600 automated reaction rate analyzer and the Cobas Bio centrifugal analysis system. Using these machines, we have established methods for the determination of proenzymes, protease-alpha 2M complexes and functional inhibitor capacities of several serine protease systems. With these automated assays a rapid system for close monitoring of the coagulation, fibrinolytic and plasma kallikrein-kinin systems in various disease states has become available.