Absorption Spectra of Normal Adults and Patients with Sickle Cell Anemia Treated with Hydrogen Peroxide at Two pH Values

The aim of the present study was application of haemoglobin absorption spectroscopy as a distinguishing tool for identification of two haemoglobin types-HbA and HbS. Millimolar absorptivities of normal adult haemoglobin (HbA) and sickle cell haemoglobin (HbS) were monitored at pH values of 7.2 and 5.0, within the ultra violet and visible spectral range (250-650 nm) in the presence of varying concentrations of hydrogen peroxide (4.00-20.00 mM). The absorption spectra of HbA and HbS exhibited the characteristic Soret band with maximum absorptivities at wavelenght (λmax) = 415 nm. Maximum absorptivities of HbA and HbS treated with H2O2 showed hypochromic red shift of the Soret band from (λmax) = 415 to 420 nm regardless of changes in pH values. In addition, increasing concentrations of H2O2 at the two pH conditions caused differential distortion and obliteration of the Soret band. Whereas pH = 7.2, [H2O2] > 14.28 mM caused obliteration of the Soret band of HbA, 20.00 mM H2O2 at lower pH = 5.0, obliterated the Soret band. In contrast to the absorption spectra of HbA, at experimental pH values of 5.0 and 7.2, maxima absorptivities of HbS were 1.8 and 2.3 mmol-1•cm-1 respectively. The spectra patterns of HbA and HbS treated with H2O2 were non-identical under the two experimental pH conditions. Therefore, these spectra absorptivity patterns can serve for the identification of HbA and HbS and to distinguish between the two haemoglobin types.