Hydrogen peroxide production from reactive liposomes encapsulating enzymes

Reactive cationic and anionic liposomes have been prepared from mixtures of dimyristoylphosphatidylcholine (DMPC) and cholesterol incorporating dimethyldioctadecylammonium bromide and DMPC incorporating phosphatidylinositol, respectively. The liposomes were prepared by the vesicle extrusion technique and had the enzymes glucose oxidase (GO) encapsulated in combination with horseradish peroxidase (HRP) or lactoperoxidase (LPO). The generation of hydrogen peroxide from the liposomes in response to externally added D-glucose substrate was monitored using a Rank electrode system polarised to +650 mV, relative to a standard silver-silver chloride electrode. The effects of encapsulated enzyme concentration, enzyme combinations (GO+HRP, GO+LPO), substrate concentration, electron donor and temperature on the production of hydrogen peroxide have been investigated. The electrode signal (peroxide production) was found to increase linearly with GO incorporation, was reduced on addition of HRP and an electron donor (o-dianisidine) and showed a maximum at the lipid chain-melting temperature from the anionic liposomes containing no cholesterol. To aid interpretation of the results, the permeability of the non-reactive substrate (methyl glucoside) across the bilayer membranes was measured. It was found that the encapsulation of the enzymes effected the permeability coefficients of methyl glucoside, increasing them in the case of anionic liposomes and decreasing them in the case of cationic liposomes. These observations are discussed in terms of enzyme bilayer interactions.