Dynamics of Hemoglobin in Human Erythrocytes and in Solution:  Influence of Viscosity Studied by Ultrafast Vibrational Echo Experiments

Ultrafast spectrally resolved stimulated vibrational echo experiments are used to measure the vibrational dephasing of the CO stretching mode of hemoglobin−CO (HbCO) inside living human erythrocytes (red blood cells), in liquid solutions, and in a glassy matrix. A method is presented to overcome the adverse impact on the vibrational echo signal from the strong light scattering caused by the cells. The results from the cytoplasmic HbCO are compared to experiments on aqueous HbCO samples prepared in different buffers, solutions containing low and high concentrations of glycerol, and in a solid trehalose matrix. Measurements are also presented that provide an accurate determination of the viscosity at the very high Hb concentration that is found inside the cells. It is demonstrated that the dynamics of the protein, as sensed by the CO ligand, are the same inside the erythrocytes and in aqueous solution and are independent of the viscosity. In solutions that are predominantly glycerol, the dynamics are modified somewhat but are still independent of viscosity. The experiments in trehalose give the dynamics at infinite viscosity and are used to separate the viscosity-dependent dynamics from the viscosity-independent dynamics. Although the HbCO dynamics are the same in the red blood cell and in the equivalent aqueous solutions, differences in the absorption spectra show that the distribution of a protein's equilibrium substates is sensitive to small pH differences.