Abstract
By using a combination of experimental neutron scattering techniques, it is possible to obtain a statistical perspective on red blood cell (RBC) shape in suspensions, and the inter-relationship with protein interactions and dynamics inside the confinement of the cell membrane. In this study, we examined the ultrastructure of RBC and protein–protein interactions of haemoglobin (Hb) in them using ultra-small-angle neutron scattering and small-angle neutron scattering (SANS). In addition, we used the neutron backscattering method to access Hb motion on the ns time scale and Å length scale. Quasi-elastic neutron scattering (QENS) experiments were performed to measure diffusive motion of Hb in RBCs and in an RBC lysate. By using QENS, we probed both internal Hb dynamics and global protein diffusion, on the accessible time scale and length scale by QENS. Shape changes of RBCs and variation of intracellular Hb concentration were induced by addition of the Na+-selective ionophore monensin and the K+-selective one, valinomycin. The experimental SANS and QENS results are discussed within the framework of crowded protein solutions, where free motion of Hb is obstructed by mutual interactions.
Highlights
Haemoglobin (Hb) is the major macromolecular fraction of red blood cells (RBCs)
By considering the short-time range probed by quasi-elastic neutron scattering (QENS), our results indicated that hydrodynamic interactions of Hb in concentrated solutions were stronger than predicted from the theory for simple colloidal hard-sphere suspensions; this predicted a value of H = 1 − 1.831φ = 0.63 for a volume fraction of φ = 0.20 being equivalent to the QENS RBC lysate sample [61]
We analysed data from Ultra-small-angle neutron scattering (USANS)/Small-angle neutron scattering (SANS) and QENS experiments that showed the influence of RBC volume on Hb–Hb interactions, and on the internal dynamics and mean diffusion coefficient of Hb in horse and human RBCs
Summary
Haemoglobin (Hb) is the major macromolecular fraction of red blood cells (RBCs). In solution, the Hb protein is a homotetramer that consists of two α- and two β-chains bearing in total four haem groups [1]. The Hb concentration in RBCs is high, with a value c = 330 mg ml−1 and a volume fraction, φ ≅ 0.25, under standard physiological conditions [2]. Due to the high volume-fraction of Hb, many other solutes are excluded from a large fraction of the cell’s cytoplasm, a phenomenon referred to as ‘macromolecular crowding’. This effect significantly increases protein folding stability, and enhances (bio)chemical reaction rates, while altering equilibrium constants of (bio)chemical reactions [3]. During RBC ageing, glucose consumption declines and RBC shape is not maintained, leading first to echinocytes (spiculated spheres) and via loss of membrane vesicles to spherocytes (smooth spheres) [10,11]. RBC morphology can be altered osmotically by increasing the K+ and Na+ concentrations in the cytoplasm, leading to swelling and dilution of the Hb therein; whereas the Hb concentration is increased in the shrunken spherocytes
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