Spectrophotometric quantification of horseradish peroxidase with o-phenylenediamine.

The assay conditions for the spectrophotometric quantification of horseradish peroxidase (HRP) with the chromogenic substrate o-phenylenediamine (OPD) at 25°C in the presence of hydrogen peroxide (H(2)O(2)) were optimized. With [OPD](0)=3.14 mM and [H(2)O(2)](0)=80 μM at pH 7.2, the initial formation of only one of the possible reaction products and intermediates, 2,3-diaminophenazine (DAP, λ(max)=417 nm), was observed. The rate of DAP formation during the first 30 min of reaction was followed spectrophotometrically and found to linearly depend on the HRP concentration between 5 and 45 pM under the conditions used.