Probing the structure-function relationship of hemoglobin in living human red blood cells.

Hemoglobin (Hb) is a key component of respiratory system and as such plays important role in human physiology. The studies of Hb's structure and functions are usually performed on cell-free protein; however, it has been shown that there are functionally relevant differences between isolated Hb and Hb present inside red blood cells (RBCs). It is clear that new experimental approaches are needed to understand the origin of these differences and to gain insight into the structure-function relationship of Hb within intact living cells. In this work we present a novel application of Resonance Raman spectroscopy to study heme active site of different forms of human Hb within living RBCs using laser excitation lines in resonance with their Soret absorption bands. These studies revealed that there are no significant changes in the disposition of the Fe-O-O fragment or the Fe-N linkage for Hb molecules enclosed in RBCs and these in free isolated states. However, some changes in the orientation of the heme vinyl groups were observed which might account for the differences in the protein activity and ligand affinity. This work highlights importance of protein-based studies and presents a new opportunity to translate these results to physiological cell systems.