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.
Fornera
Sara
S
Department of Materials, ETH Zürich, CH-8093 Zürich, Switzerland.
Walde
Peter
P
D006735:Horseradish Peroxidase / Q000032:analysis*; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D010619:Phenazines / Q000378:metabolism; D010655:Phenylenediamines / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods*
D010619:Phenazines; D010655:Phenylenediamines; C054312:2,3-diaminophenazine; C034193:1,2-diaminobenzene; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase