Spectrophotometric Quantification of Peroxidase with p-Phenylene-diamine for Analyzing Peroxidase-Encapsulating Lipid Vesicles

A spectrophotometric assay for the determination of horseradish peroxidase (HRP) in aqueous solution with p-phenylenediamine (PPD, benzene-1,4-diamine) as electron donor substrate and hydrogen peroxide (H2O2) as oxidant was developed. The oxidation of PPD by HRP/H2O2 leads to the formation of Bandrowski’s base ((3E,6E)-3,6-bis[(4-aminophenyl)imino]cyclohexa-1,4-diene-1,4-diamine), which can be quantified by following the increase in absorbance at 500 nm. The assay was applied for monitoring the activity of HRP inside ≈180 nm-sized lipid vesicles (liposomes), prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and purified by size exclusion chromatography. Because of the high POPC bilayer permeability of PPD and H2O2, the HRP-catalyzed oxidation of PPD occurs inside the vesicles once PPD and H2O2 are added to the vesicle suspension. In contrast, if instead of PPD the bilayer-impermeable substrate ABTS2– (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) is used, the oxidation of ABTS2– ...