SELECTIVE MONITORING OF PEPTIDASE ACTIVITIES WITH SYNTHETIC POLYPEPTIDE SUBSTRATES AND POLYION-SENSITIVE MEMBRANE-ELECTRODE DETECTION

A novel method to monitor specific peptidase activities in biological samples as complex as undiluted plasma/blood is described. The approac h is based on the design of synthetic polypeptide substrates in which di- or triarginine sequences are linked to each, other via one or more other amino acids recognized specifically by the peptidase to be dete rmined. Detection of chymotrypsin and renin activities using synthetic substrates P4 (F-R-R-R-F-V-R-R-F-NH2) and P5 (R-R-R-L-L-R-R-L-L-R-R-R ), respectively, serves to demonstrate the principles of this new assa y system. A polyion-sensitive membrane electrode, prepared by doping p olymer films with dinonyhlaphthalene-sulfonate (DNNS), is shown to exh ibit significant nonequilibrium electromotive force (EMF) responses to ward these and other polycationic substrates at er levels under physio logical conditions: The same electrode, however, exhibits much smaller total EMF response toward the shorter fragments of the synthetic pept ides generated by peptidase activity; hence, the addition of peptidase to a solution containing the synthetic substrate yields a change in e lectrode EMF response, the rate of which is proportional to the activi ty of peptidase present. Other synthetic polycationic peptides as ell as natural polycationic peptides (e.,., protamine) that lack specific cleavage sites for chymotrypsin and renin, yet are detected by the DNN S-based membrane electrode, do not elicit any significant change in EM F response in the presence of the peptidases, confirming the feasibili ty and utility of the proposed bioanalytical method.