Evidence for a potential-dependent reversible inactivation of urease adsorbed on a gold electrode

Abstract

The potentiometric collection mode of the scanning electrochemical microscope has been applied to a kinetic study of changes in immobilised enzyme activity. Urease adsorbs spontaneously onto gold surfaces from aqueous solution and the immobilised enzyme was shown to retain activity as a catalyst for the hydrolysis of urea to ammonium and bicarbonate ions. Urease activity was monitored using an ion-selective potentiometric tip electrode to detect a product (NH4+) of the enzymatic reaction and the use of a 50 µm radius gold electrode to immobilise the enzyme results in a steady state diffusion field for the reaction products and enables quantitative interpretation of the data. The ammonium ion flux determined from the tip potential decreased by approximately 40% when the potential of the gold was scanned or stepped across the range from 0.0 to+0.4 V vs. Ag/AgCl. The decrease in flux is reversible and the original value almost completely recovered after the potential was returned to 0 V. The flux decrease is attributed to inactivation of the enzyme. The timescale of the inactivation is limited by the establishment of a steady state concentration profile of ammonium ions at the 50 µm radius source. The kinetics of the reactivation are significantly slower with an apparent first order rate constant of 0.022 s-1. No faradaic processes were observed at the gold electrode over the potential range from 0.0 to +0.4 V. We therefore suggest that changes in the enzyme quaternary structure due to the changing electric field and ionic composition at the interface are responsible.