Scalar couplings as pH probes in compartmentalized biological systems: 31P NMR of phosphite.

The use of scalar couplings in nuclear magnetic resonance (NMR) spectra was investigated as a possible tool for the measurement of pH in different compartments of biological systems. The proposed method is attractive because no internal reference is required, unlike more widely used chemical shift titrations. The phosphite anion is shown to be ideal for the measurement of pH in the physiological range. In isotonic solution, the divalent anion PHO(3)(2-) has a one-bond (1)J(PH) of 568.1 Hz, increasing to 620.7 Hz for the monovalent anion PH(OH)O(2)(-), with a measured pK(a) of 6.19. The technique was applied to the measurement of pH in a suspension of human erythrocytes. The (31)P spectra of these species give well-resolved doublets for the intra- and extracellular spaces, and can be used to measure the pH difference across the cell membrane with errors on the order of about 0.01 pH units in the range of pH 5-7. Variations in erythrocyte pH due to metabolic changes are clearly observed. Copyright 2003 Wiley-Liss, Inc.