³¹P NMR measurements of intracellular pH in erythrocytes: Direct comparison with measurements using freeze‐thaw and investigation into the influence of ionic strength and Mg²⁺

Abstract

The pH-dependent chemical shift behavior in 31P NMR spectroscopy of inorganic phosphate (Pi) and methylphosphonic acid (MeP) in solutions of various composition at 37 degrees C are presented. An ionic strength dependent lowering of the pKa values of both Pi and MeP is observed. 3 mmol/liter Mg2+ did not affect the pH-dependent chemical shift behavior of Pi and MeP in solutions containing 135 mmol/liter KCl and 5 mmol/liter NaCl. This finding was reinforced by adding 5 mmol/liter 2,3-diphosphoglycerate (2,3-DPG) and 1.5 mmol/liter ATP to solutions with 3 mmol/liter Mg2+. The presence of these Mg2+-binding substances had no detectable effect on the chemical shift of Pi and MeP at various pH values. The pH dependence of the chemical shift of MeP, Pi, and 2,3-DPG in oxygenated erythrolysates was compared to protein free model solutions. No significant differences could be detected for MeP and Pi whereas there was a change in the chemical shift of about 0.1-0.2 ppm for both phosphates in 2,3-DPG. Finally intracellular pH estimated by 31P NMR were compared to pH measured by electrode in freeze-thawed hemolysates. At an extracellular pH of 7.341 +/- 0.045 (SD) (n = 6) the intracellular pH was 7.160 +/- 0.014 (SD) by the freeze-thaw procedure and 7.213 +/- 0.027 (SD), 7.172 +/- 0.029 (SD), and 7.152 +/- 0.033 (SD) estimated by the chemical shift of MeP, and 3-phosphorus and 2-phosphorus of 2,3-DPG, respectively.