A resonance Raman spectroscopic investigation into the effects of fixation and dehydration on heme environment of hemoglobin

Abstract

The effects of fixation and dehydration procedures on heme environment inside human erythrocytes were examined using resonance Raman spectroscopy. The resonance Raman spectroscopic data along with far-infrared spectra show that hemoglobin in air-dried red blood cells and those fixed in the precipitating fixatives, methanol and acetone, changes to hemichrome, a low-spin component in which the sixth coordination site of the iron is occupied by the imidazole group of the distal histidine. The cross-linking fixatives, formaldehyde and glutaraldehyde, maintain hemoglobin in its native conformational state (oxyhemoglobin) as evidenced by the appearance of the oxygen ligand marker bands along with the skeletal sensitive modes. However, polymerization with cross-linking fixatives increases the auto-oxidation kinetics of hemoglobin and gradually oxidizes it to the met state. Moreover, the dehydration of a red blood cell under N₂ results in a change of oxyhemoglobin to a six coordinate low-spin Fe^II derivative of hemoglobin, hemochrome. Copyright © 2009 John Wiley & Sons, Ltd.