HR‐MAS of cells: A “cellular water shift” due to water‐protein interactions?

Abstract

Under HR-MAS conditions, cells are subjected to high centrifugal forces that may cause irreversible cell damage. First, conditions have been defined to monitor and keep to a minimum unwanted effects in HR-MAS spectra arising from the loss of cell integrity. Then, the HR-MAS spectra of reasonably intact cells have been analyzed. Cell suspensions subjected to MAS rates as low as 1 kHz split into a two-compartment system that is composed of a cell-rich phase (H 2 O i ) and a cell-free phase (H 2 O o ). Each of these phases is characterized by its own water 1 H-NMR signal. Transport of water molecules between the cellrich and cell-free compartments is limited by the very low contact area between the two compartments, and water exchange dynamics consequently fall into the slow exchange limit on the NMR timescale. Since the exchange between the two water populations is "frozen,

Water chemical shift in 1H NMR of red cells: Effects of pH when transmembrane magnetic susceptibility differences are low

“…When Δχ is small (<10% of χ), the intracellular protein concentration is the primary determinant of the extent of splitting between the intracellular and supernatant 1 H 2 O resonances (2, 9). Since hemoglobin is by far (>95%) the predominant protein in RBCs (15), the magnitudes of the splitting determined for RBCs where the suspension pH or osmolality was altered, or from different donors, were compared with the mean corpuscular hemoglobin concentrations (MCHCs) of the RBCs.…”

“…Two resonances are observed for 1 H 2 O in magic angle spinning (MAS) NMR spectra of erythrocyte (red blood cell; RBC) and other cellular suspensions (1–3). The signals are separated by ≈18 Hz (Fig.…”

“…For cylindrical samples oriented at the magic angle there is no contribution to the splitting of the water peak from a magnetic susceptibility difference between the cells and the suspension medium (3, 7). Separate intracellular and supernatant resonances are also seen in 31 P spectra of phosphoryl compounds in RBC suspensions (8), where it has been shown that the separation is brought about through differences in the extent of hydrogen bonding to the phosphoryl oxygen due to the high protein concentration in RBCs, the so‐called “hydrogen‐bond split‐peak effect.” In the case of water in MAS experiments, the “splitting” also represents a true chemical shift difference between water in the two compartments, brought about by differences in the extent of hydrogen bonding of water, with the magnitude of the separation being dependent on the intracellular protein concentration, and not the pH of the suspension medium (2, 9).…”

Water exchange across the erythrocyte plasma membrane studied by HR‐MAS NMR spectroscopy

“…An alternative explanation for a single water signal in immobilized erythrocytes is a loss of membrane integrity due to the chemical/physical treatment used to immobilize the RBCs. If RBC plasma membranes are disrupted, the magnetic diversity between intra‐ and extracellular water molecules is abolished, leading to a single pool of water with spectral parameters that are the average between those of intra‐ and extracellular water (15). To assess the integrity of the plasma membranes in immobilized RBCs, we resorted to 19 F‐NMR spectroscopy, as described in the next section.…”

“…1). Transport of water molecules between the cell‐rich (H 2 O pellet ) and cell‐free (H 2 O super ) compartments is limited by the very low contact area between these compartments (13, 15). The rate‐determining step for the exchange between the water pools giving rise to the two 1 H‐NMR signals is not the transport across the plasma membrane; rather, it is represented by diffusion of water molecules from the bulk of H 2 O pellet phase and the bulk of H 2 O super phase.…”