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) ba sed on its enzymatic activity for the oxidation of o-phenylenediamine with hydrogen peroxide. Stopped-flow spectrophotometry was used to study the kin etic behavior of the oxidation reaction. The absorbance of the reaction pro duct, 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 degrees C was proportional to the concentration of Hb i n the range of 8.5 x 10(-5)6.5 x 10(-11) mol/l. Excess of bovine serum albu min, Ca(II), Mg(II), Cu(II) and glucose did not interfere. The relative sta ndard deviations were within 5% for the determination of different concentr ations of Hb. The detection limit was found to be 3.2x10(-12) mol/l. (C) 20 00 Elsevier Science B.V. All rights reserved.