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+

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Abstract
The pH‐dependent chemical shift behavior in 31P NMR spectroscopy of inorganic phosphate (P1) and methylphosphonic acid (MeP) in solutions of various composition at 37°C are presented. An ionic strength dependent lowering of the pKa. values of both P1 and MeP is observed. 3 mmol/liter Mg2+ did not affect the pH‐dependent chemical shift behavior of P1 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 Mg+‐binding substances had no detectable effect on the chemical shift of P1 and MeP at various pH values The pH dependence of the chemical shift of MeP, P1 and 2,3‐DPG in oxygenated erythrolysates was compared to protein free model solutions. No significant differences could be detected for MeP and P1 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. © 1987 Academic Press, Inc.