The hemoglobin concentration of 35 g/dl of human red blood cells is close to the solubility threshold. Using microwave dielectric spectroscopy, we have assessed the amount of water associated with hydration shells of methemoglobin as a function of its concentration in the presence or absence of ions. We estimated water-hemoglobin interactions to interpret the obtained data. Within the concentration range of 5-10 g/dl of methemoglobin, ions play an important role in defining the free-to-bound water ratio competing with hemoglobin to recruit water molecules for the hydration shell. At higher concentrations, hemoglobin is a major contributor to the recruitment of water to its hydration shell. Furthermore, the amount of bound water does not change as the hemoglobin concentration is increased from 15 to 30 g/dl, remaining at the level of ∼20% of the total intracellular water pool. The theoretical evaluation of the ratio of free and bound water for the hemoglobin concentration in the absence of ions corresponds with the experimental results and shows that the methemoglobin molecule binds about 1400 water molecules. These observations suggest that within the concentration range close to the physiological one, hemoglobin molecules are so close to each other that their hydration shells interact. In this case, the orientation of the hemoglobin molecules is most likely not stochastic, but rather supports partial neutralization of positive and negative charges at the protein surface. Furthermore, deformation of the red blood cell shape results in the rearrangement of these structures.
Bibliographical noteFunding Information:
We would like to thank Keysight Technologies Israel Ltd. and INTERLLIGENT RF & Microwave Solutions for the loan of the Vector Network Analyzer Agilent N5234B PNA-L. We would also like to thank the Swiss National Science Foundation CRSII5_180234 for their financial support for the research project “Premembrane and cytosolic water as a marker of red blood cell aging in vivo and in vitro.” The modeling study (Figs. 2 and 8–11) was supported by the Russian Science Foundation, Grant No. 19-14-00374. We would also like to thank ISF 341/18 for their financial support for this project. There are no conflicts to declare.
© 2021 Author(s).