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 degrees C in the presence of hydrogen peroxide (H2O2) were optimized. With [OPD](0) = 3.14 mM and [H2O2](0) = 80 mu M at pH 7.2, the initial formation of only one of the possible reaction products and intermediates, 2,3-diaminophenazine (DAP, lambda(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. (C) 2010 Elsevier Inc. All rights reserved.