Preferential role of iron in heme degradation of hemoglobin upon gamma irradiation

It is usually believed that γ-ray interaction with biomolecules is intermediately performed by reactive oxygen species (ROS) produced from radiolysis of water. Hemoglobin (Hb) as one of the most abundant biomolecule in blood and well-studied endogenously affected by ROS, was a good candidate for study. Adult human Hb was extracted and irradiated using four distinct 20, 60, 90 and 170Gy doses from Co-60 γ-ray source. UV-vis, fluorescence and FT-IR spectroscopies were used to study the whole conformational changes and partial degradation of heme. Hb species calculated using Benesch equations indicated that the concentration of oxy-Hb was decreased from 9.97μM to 6.56μM, while the total metastable met and deoxy-Hb concentration were just increased 2.39μM and about 8.4% of total heme was diminished. Heme degradation was studied using fluorescence spectra at two 321 and 460nm excitation wavelengths as fully and partially degradation of heme respectively. Inverse behavior of these two fluorescence spectra suggested a new mechanism of heme degradation in which γ-ray preferably absorbed by heme without any intermediary effects of water. It was confirmed by FT-IR spectra at 900-1000cm-1 where the FeN and NH of porphyrin indicate their own stretching vibrational bands. Thermal stability justified that the gamma radiation induced the conformational changes of Hb which is appeared during thermal unfolding. First derivative of thermal spectra indicated that the Tm of 170Gy dose irradiated sample is 2°C lowered and total concentration of Hb was decreased 14%.