Assay of H2O2 by HRP catalysed co-oxidation of phenol-4-sulphonic acid and 4-aminoantipyrine: characterisation and optimisation

A colorimetric system involving horseradish peroxidase (HRP), phenol-4-sulfonic acid (PSA) and 4-aminoantipyrine (4-AAP) for the determination of hydrogen peroxide concentration and peroxidase activity, and possible coupling with oxidases for other analytical purposes, was developed and optimised. Proposed reagents concentrations are 25 mM PSA, 0.4 mM 4-AAP, and 100 mM phosphate buffer pH 7. The absorptivity of the product calculated to the added hydrogen peroxide concentration is close to value measured for the colorimetric system that instead of PSA contained phenol. Influence of some common fermentation broth components (d-galactose, d-glucose, succinate, glycerol, acetate, yeast extract) on the colorimetric system was tested and no significant interference was found, except for yeast extract for concentrations above 5 g l−1. Some advantages of the proposed colorimetric system is that PSA has better solubility, does not produce precipitates in reaction with hydrogen peroxide, and is not as toxic as phenol. The optimised colorimetric system was used in soluble and immobilised HRP kinetics experiments. Optimised colorimetric system was also successfully employed in a flow injection analysis system for hydrogen peroxide measurements and good linear range, sensitivity, reproducibility and system stability were confirmed.