Micro-Raman Spectroscopy of the Hemoglobin Iron-Histidine Bond in Single Erythrocytes

Abstract

The iron-histidine vibrational mode probes the crucial iron-protein linkage in heme proteins, and it has been established as a sensitive structural probe of the proximal heme pocket from studies of proteins in solution. The cellular environment can modify the interactions and dynamics from the isolated protein. We present Raman measurements on individual erythrocytes under physiological conditions over the frequency range from 150 to 1700 cm-1. Raman spectra were excited with the 442 nm line from a He-Cd laser using 2-3 mW power at the sample. Photodissociation of the ligated hemoglobin is achieved by the excitation laser beam and monitored by the oxidation state marker bands. We investigate the pressure dependence of the iron-histidine stretching mode and compare with results on isolated hemoglobin and myoglobin. A pressure dependent shift suggests a conformational change of the heme environment with pressure.