ELECTROCHEMICAL ASSAY OF PROTEINASE-INHIBITORS USING POLYCATION-SENSITIVE MEMBRANE-ELECTRODE DETECTION

A simple and sensitive electrochemical method suitable for real time d etection of trypsin-like proteinase inhibitors is described. The metho d is based, on utilizing a protamine (a polycationic substrate for try psin-like proteinases)-sensitive membrane electrode to monitor, potent iometrically, the initial rate of protamine decomposition upon the add ition of a proteinase-antiproteinase test solution. In the presence of proteinase inhibitors, the initial rate of change in electromotive fo rce is dependent on the concentration of proteinase inhibitor in the s ample solution. The feasibility of this new assay method is demonstrat ed by detecting four inhibitors of trypsin-like proteinases: alpha(1)- antiproteinase inhibitor, alpha(2)-macroglobulin, aprotinin, and soybe an inhibitor, using trypsin as the indicator proteinase. The efficacy of inhibition by each species, as expressed by I-50 values (concentrat ion of the inhibitor that induces 50% of the maximum proteinase inhibi tion), is shown to correlate well with literature values for the assoc iation constant of the proteinase-antiproteinase complex (k(assoc)). T he proposed electrochemical assay for aprotinin is examined further us ing trypsin, plasmin, and kallikrein as the proteinase indicator reage nts, It is found that the trypsin-aprotinin system offers the highest sensitivity and lowest detection limit for aprotinin detection. Applic ation of the proposed method for measuring aprotinin in pretreated pla sma samples is also reported. (C) 1997 Academic Press.