[show abstract][hide abstract] ABSTRACT: The hydrogen donor couples pyrocatechol-aniline and phenol-aminoantipyrine in the presence of hydrogen peroxide were compared as chromogens for horseradish peroxidase (HRP) assay. UV-Visible spectroscopy and high-performance liquid chromatography analysis indicated that during the HRP biocatalytic process, pyrocatechol-aniline was converted to a pink-colored reagent with a lambda(max) of 510 nm, which was used in the assay of HRP activity. Electrochemical studies revealed adequate electron transfer ability for this color reagent to serve as a proper mediator for HRP also. Using pyrocatechol-aniline a higher sensitivity and lower detection limit was obtained relative to those of the phenol-aminoantipyrine couple, which is commonly used for HRP assay. A relative standard deviation of 2.9% was obtained for 20 HRP activity measurements, indicating a satisfactory reproducibility for this method. In addition, kinetic parameters of K(m) (12.5mM) and V(max) (12.2 mM min(-1)mg(-1)) were calculated for pyrocatechol-aniline. Regarding the superiority of pyrocatechol-aniline, this couple is suggested to be a better hydrogen donor for the HRP spectrophotometric assay.
[show abstract][hide abstract] ABSTRACT: Direct electron transfer of horseradish peroxidase, immobilized on a functional membrane-modified gold electrode, was studied. The electrode showed a quasi-reversible electrochemical redox behavior with a formal potential of 60mV (versus Ag/AgCl) in 20mM potassium phosphate buffer solution at pH 7.0 and temperature 25°C. The cathodic transfer coefficient was 0.42 and electron transfer rate constant was evaluated to be 1.6s−1. Furthermore, the modified electrode was used as a biosensor and exhibited a satisfactory stability and sensitivity to H2O2. The linear range of this biosensor for H2O2 determination was from 5.0×10−6 to 1.5×10−4M while its detection limit, based on a signal-to-noise ratio of 3, was 1.3×10−6M. The apparent Michaelis–Menten constant (Kmapp) for immobilized HRP was calculated to be 1.6×10−4M.