Determination of hemoglobin based on its enzymatic activity for the oxidation of o-phenylenediamine with hydrogen peroxide

A new method has been developed for the determination of hemoglobin (Hb) based on its enzymatic activity for the oxidation of o -phenylenediamine with hydrogen peroxide. Stopped-flow spectrophotometry was used to study the kinetic behavior of the oxidation reaction. The absorbance of the reaction product, 2,3-diaminophenazine, at the wavelength of 425 nm was recorded every millisecond during the first 0.2 min of the reaction. The initial reaction rate obtained at 48°C was proportional to the concentration of Hb in the range of 8.5×10 −5 –6.5×10 −11 mol/l. Excess of bovine serum albumin, Ca(II), Mg(II), Cu(II) and glucose did not interfere. The relative standard deviations were within 5% for the determination of different concentrations of Hb. The detection limit was found to be 3.2×10 −12  mol/l.