Characterization of transmembrane chemical shift differences in the 31P NMR spectra of various phosphoryl compounds added to erythrocyte suspensions

Trimethyl phosphate, dimethyl methylphosphonate, diethyl methylphosphonate, trimethylphosphine oxide, and the hypophosphite, phenylphosphinate, and diphenylphosphinate ions all contain the phosphoryl functional group. When added to an intact erythrocyte suspension at 20 degrees C, each of the compounds gave rise to separate intra- and extracellular 31P NMR resonances, and the separation between the two resonances of each compound varied with the mean cell volume. The differences between the intra- and extracellular chemical shifts were shown to be primarily attributable to the effects of hemoglobin. The presence of hemoglobin inside the cell gave rise to a significant difference in the magnetic susceptibilities of the two compartments. In addition, it exerted a large susceptibility-independent chemical shift effect, the magnitude of which was dependent upon the chemical structure of the phosphoryl compound involved. A number of other intra- and extracellular components were also shown to cause chemical shift variations, smaller than those arising from hemoglobin but nonetheless significant. The cell volume dependence of the transmembrane chemical shift differences therefore reflected not only the cell volume dependence of the intracellular hemoglobin concentration but also the changing concentration of the other solutes in the two compartments. In addition to their cell volume dependence, the transmembrane chemical shift differences varied with temperature. In the case of the nonelectrolytes this reflected not only the temperature dependence of the mechanism(s) responsible for the susceptibility-independent shift effects but also the temperature dependence of the rates at which the compounds traversed the cell membrane.