Abstract

The distribution and conformational state of hemoglobin (Hb) within erythrocytes are critical factors influencing red blood cell (RBC) function and oxygen transport efficiency. This study investigates the regulatory role of extracellular calcium ions ([Ca²⁺]out) on the biophysical properties of the erythrocyte membrane and the associated Hb. Using a combination of spectroscopic and biophysical techniques, we demonstrate that a decrease in [Ca²⁺]out significantly alters the erythrocyte surface charge, leading to an increase in the membrane's zeta-potential. This change in the membrane environment is directly correlated with a redistribution of Hb, specifically a decrease in the content of membrane-associated Hb and a change in its intracellular localization. Furthermore, the reduction in [Ca²⁺]out induces distinct conformational changes in both the heme and globin components of the Hb molecule, evidenced by alterations in the vibrational spectra of the pyrrole semirings and vinyl groups, and a decrease in the rigidity of the heme's protein surrounding. These findings suggest that extracellular Ca²⁺ acts as a crucial external modulator, linking the external environment to the internal molecular organization of the RBC, with implications for understanding erythrocyte stability and the pathogenesis of various hematological disorders.