Microviscosity of human erythrocytes studied with hypophosphite and 31P-NMR.

A 31P-NMR method, which complements earlier 13C-NMR procedures for probing the intra-erythrocyte microenvironment, is described. Hypophosphite is an almost unique probe of the erythrocyte microenvironment, since it is rapidly transported into the cell via the band 3 protein, and intra- and extracellular populations give rise to distinct resonances in the 31P-NMR spectrum. Relaxation mechanisms of the 31P nucleus in the hypophosphite ion were shown to be spin-rotation and dipole-dipole. Analysis of longitudinal relaxation rates in human erythrocytes, haemolysates and concentrated glycerol solutions allowed the determination of microviscosity using the Debye equation. Bulk viscosities of lysates and glycerol solutions were measured using Ostwald capillary viscometry. Translational diffusion coefficients were then calculated from the viscosity estimates using the Stokes-Einstein equation. The results with a range of solvent systems showed that 'viscosity' is a relative phenomenon and that bulk (i.e., macro-) viscosity is therefore not necessarily related to the NMR-determined viscosity. The intracellular NMR-determined viscosities from red cells, ranging in volume from 65.5 to 100.1 fl, varied from 2.10 to 2.67 mPa s. This is consistent with the translational diffusion coefficients of the hypophosphite ion altering by only 20%, whereas the values determined from bulk viscosity measurements conducted on lysates of these cells are consistent with a 230% change.

Association of ferri- and ferro-cytochrome c with lipid multilayers: a 31P solid-state NMR study.

The 31P nuclear magnetic resonance anisotropies of dispersions of diacylphosphatidic acid and diacylphosphatidylserine were slightly increased in the presence of cytochrome c: no increase was observed with cardiolipin. However, the 31P spin-lattice relaxation times (T1) for all of these lipids were reduced markedly by the protein. As similar effects were observed with ferri-cytochrome c and with the reduced protein, which is diamagnetic, we suggest that the changes in T1 reflect a reduction in the spectral density of fast motions for the lipid headgroups attendant on binding of protein, rather than paramagnetic relaxation of the phosphorus nuclear spin.