Resonance Raman spectroscopy provides evidence of heme ordering within the functional erythrocyte

Resonance Raman spectra of functional erythrocytes aligned in vertical and horizontal orientations relative to the optical path are presented. Dramatic differences were observed between the two orientations as demonstrated by comparing mean spectra recorded from 30 cells in each orientation and analyzing the results with principal components analysis (PCA). In particular, bands at 1605, 1561, 1546, 1338, 1305 and 1227 cm−1 are more intense in the vertical orientated cells, while bands at 1617, 1118 and 997 cm−1 are more intense in the horizontally orientated cells. Depolarization ratios calculated for each cell orientation show that the vertical orientated cells exhibit more polarized bands, while the horizontal orientated exhibit more anomalously polarized bands. The results indicate that a distinct ordering of hemes exists in the functional red blood cell. The polarization measurements reported herein provide further evidence that significant ordering exists in the red cell. It is postulated that heme ordering within the functional erythrocyte could facilitate oxygen and/or energy transfer in the macromolecular crowded environment of this highly energetic cell.