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

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 ABTS^2- (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) is used, the oxidation of ABTS^2- inside the vesicles does not occur. Therefore, using PPD and ABTS^2- in separate assays allows distinguishing between vesicle-trapped HRP and HRP in the external bulk solution. In this way, the storage stability of HRP-containing POPC vesicles was investigated in terms of HRP leakage and activity of entrapped HRP. It was found that pH 7.0 suspensions of POPC vesicles (2.2 mM POPC) containing on average about 12 HRP molecules per vesicle are stable for at least 1 month without any significant HRP leakage, if stored at 4 °C. Such high stability is beneficial not only for bioanalytical applications but also for exploring the kinetic properties of vesicle-entrapped HRP through simple spectrophotometric absorption measurements with PPD as a sensitive and cheap substrate.