The electrochemical activity determination of trypsin-like enzymes I - trypsin

Since 1970, a number of chromogenic substrates have been synthesized for the assay of the activities of enzymes intervening along the blood clotting process. The related studies have in all cases been restricted to homogeneous media. The aim of this work is to establish the conditions for the determination of enzymatic activities in the presence of solid dispersions. The principle of this analytical technique is based on the electrochemical detection of electroactive species, which can be directly related to the extent of an enzyme-catalyzed hydrolysis. For that purpose, a new substrate, benzoyl D,L arginyl p-aminodiphenylamide hydrochloride ( D,L BAPADA) has been synthesized and the method has been tested with trypsin as the enzyme. The substrate proved to be specific toward this enzyme. The amine released during the hydrolysis, the p-aminodiphenylamine (pADA), is electroactive in the anodic potential range, where neither the reduction of oxygen, nor the background discharge interfere. The amount of amine is periodically detected by its oxidation peak produced with an adequate potentiodynamic signal. The effects of several parameters which may affect the response signal have been checked by comparison with the calibration curve of the amine traced in the conditions met for the assay. Although the oxidation peak of the amine is qualitatively affected, the amperometric procedure nevertheless allows to use the calibration curve in order to convert the currents in terms of concentrations.By the use of a pretreated platinum electrode, first-order kinetics were obtained for the trypsin-substrate system. The magnitude of the kinetic constants fairly well compares with the spectrophotometric experiments carried out with substrates of the same kind.