Horseradish peroxidase (HRP, E.C.1.11.1.7) is a secretory enzyme that in vivo takes part in the formation of free radicals vital in polymerisation reactions and in vitro catalyses the oxidation of a wide variety of substrates including phenols, aromatic amines, thioanisoles and iodide, by hydrogen peroxide.

Enzymatic catalysis in organic solvents is being increasingly used in a wide variety of processes such as enzymatic analysis, polymerisation and biotransformations.

The development of a complete organic assay was not possible since hydrogen peroxide was not soluble in the hydrophobic organic solvents studied. To cope with this problem we used reversed micelles as carriers of the hydrogen peroxide, while the phenolic compounds were dissolved in the organic solvent.

The peroxidative activity of HRP solubilized in AOT (di[2-ethylhexyl] sulfosuccinate) reversed micelles in isooctane and decalin has been investigated using the substrate guaiacol (2-metoxyphenol) which is soluble in aqueous and organic solvents.

The activity of the enzyme in both solvents was determined as a function of the water content, which was defined by the parameter w₀ (molar ratio between water and AOT concentration). These profiles, unlike those which most enzymes exhibit, are not bell shaped since the activity does not decrease after reaching a maximum value but remains fairly constant. The effect of pH in the activity profile was also studied. The enzyme is fully active at pH 7 and 8 for water contents higher than 3.5% (v/v) but completly deactivates at pH 9.

The stability of HRP is strongly dependent on the water content of the system. Higher levels of stability were obtained for higher values of w₀. HRP stability is also affected by the presence of substrates. Whilst the stability increases markedly when the enzyme is incubated with guaiacol, it does not appear to be so strongly affected by the presence of hydrogen peroxide, at the concentrations studied.