The electrode adsorption method for determination of enzyme activity: a study of substrate requirements

Abstract

The electrode adsorption method for the determination of enzyme activity requires substrates that, besides having good kinetics constants for the enzyme, also show good adsorption/desorption kinetics to the electrode surface and adsorb in such a way that they change the double-layer capacitance of the electrode. A series of peptide substrates containing one to three aromatic groups has been synthesized. Our results show that the aromatic groups are of crucial importance for the capacitance change caused by the adsorbing/desorbing substrate. Thus, the tripeptide substrate, Bz-Phe(NO2)-Val-Arg-pNA, with three aromatic groups is superior to the other synthesized substrates containing only one or two aromatic groups. Our desorption experiments show that several factors determine the rate of capacitance increase observed when thrombin is added to a substrate solution in equilibrium with a substrate-covered electrode. The kinetic constants of the substrate determine how the substrate concentration in the solution decreases and, consequently, determine the spontaneous desorption measured as capacitance increase. Thrombin does not seem to split adsorbed substrate molecules but it adsorbs to the substrate-covered surface and in that way causes a capacitance decrease counteracting the change caused by desorption of substrate.