Phospholipase A2 activity on supported thiolipid monolayers monitored by electrochemical and SPR methods

Abstract

A lipolytic enzyme, pork pancreatic phospholipase A2, was used as a tool for modifying in a controlled way the structure of a supported thiolipid (1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol) film. The electrode covered with a self-assembled monolayer of the thiolipid was immersed in the phospholipase aqueous solution to allow adsorption of the enzyme and catalytic hydrolysis to occur. Phospholipase catalyzed the cleavage of the ester bond in the sn-2 position of the phospholipids attached to the electrode and thus created defects in the thiolipid film. Surface plasmon resonance was used for monitoring of the process, namely enzyme adsorption, phospholipid hydrolysis and product desorption; Triton X-100 solution removed the lipolysis products from the electrode. Cyclic voltammetry and electrochemical impedance spectroscopy measurements show that the efficiency of the transport of an electroactive probe, ferrocyanate, and of an electroactive drug, doxorubicin, through the film, depends on the action of the enzyme. Indeed, the latter controls the state and the electrical properties of the lipid layer covering the electrode surface. The doxorubicin reduction/oxidation signals appearing at potentials close to those observed using bare gold electrode indicated a facilitated penetration of the drug through the monolayer due to formation of pore-like defects by phospholipase A2.