Water exchange across the plasma membrane of erythrocytes (red blood cells (RBCs)) was studied by means of high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. Under HR-MAS conditions, the centrifugal force causes the splitting of RBC suspensions into a two-phase system composed of a central core of cell free water and an outer layer of tightly packed cells. Water belonging to each of these phases gives rise to two separated resonances. Chemical exchange between them is not detectable on the chemical shift or saturation transfer (ST) NMR time scale because of the physical separation between the phases. When the RBCs are dispersed and immobilized within a matrix made of cross-linked albumin, the splitting into a two-phase system is prevented and a single exchange-averaged peak for water is detected in H-1 HR-MAS NMR spectra. The lineshape of this peak is dependent on transmembrane exchange kinetics, since MAS averages out all the anisotropic magnetic interactions that are responsible for additional line-broadening under conventional liquid conditions. Line-shape analysis according to a two-site exchange model yielded a residence lifetime on the order of about 10 ms (at 37 degrees C) for a water molecule within the intracellular compartment, which is not too far from the generally accepted value of 9.6-14.8 ms.
Water exchange across the erythrocyte plasma membrane studied by HR-MAS NMR spectroscopy
BRUNO, ERIK;BARONI, SIMONA;AIME, Silvio
2006-01-01
Abstract
Water exchange across the plasma membrane of erythrocytes (red blood cells (RBCs)) was studied by means of high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. Under HR-MAS conditions, the centrifugal force causes the splitting of RBC suspensions into a two-phase system composed of a central core of cell free water and an outer layer of tightly packed cells. Water belonging to each of these phases gives rise to two separated resonances. Chemical exchange between them is not detectable on the chemical shift or saturation transfer (ST) NMR time scale because of the physical separation between the phases. When the RBCs are dispersed and immobilized within a matrix made of cross-linked albumin, the splitting into a two-phase system is prevented and a single exchange-averaged peak for water is detected in H-1 HR-MAS NMR spectra. The lineshape of this peak is dependent on transmembrane exchange kinetics, since MAS averages out all the anisotropic magnetic interactions that are responsible for additional line-broadening under conventional liquid conditions. Line-shape analysis according to a two-site exchange model yielded a residence lifetime on the order of about 10 ms (at 37 degrees C) for a water molecule within the intracellular compartment, which is not too far from the generally accepted value of 9.6-14.8 ms.
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