Regulation of Intracellular pH by Human Peripheral Blood Lymphocytes as Measured by 19F NMR
Abstract
We have measured the intracellular pH of human peripheral blood lymphocytes by means of high-resolution 19F NMR spectroscopy using D,L-2-amino-3,3-difluoro-2-methylpropanoic acid (F2MeAla) as a probe. Lymphocytes readily took up the methyl ester of F2MeAla, and endogenous esterase hydrolyzed the ester to the free amino acid inside the cell. This alpha-methyl amino acid is not metabolized by the cell, and its 19F NMR spectrum exhibits large pH-dependent shifts as the alpha-amino group is protonated. The size of the 19F shifts, the high sensitivity of 19F NMR, and the favorable pKa of the alpha-amino group of F2MeAla (pKa = 7.3) allowed us to measure intracellular pH of lymphocytes at 25-30 degrees C with approximately 5-min acquisition times. Measurements at various external pH values demonstrated that human peripheral blood lymphocytes regulate their internal pH, a process requiring expenditure of metabolic energy. In the pH range between 6.8 and 7.4, lymphocytes maintain a constant internal pH of 7.17 +/- 0.06 pH unit. Outside this range, intracellular pH changes with extracellular pH. The accuracy of this 19F pH probe has been confirmed by independent measurements of intracellular pH using equilibrium distributions of 5,5-dimethyloxazolidine-2,4-dione.
- Publication:
-
Proceedings of the National Academy of Science
- Pub Date:
- December 1982
- DOI:
- 10.1073/pnas.79.24.7944
- Bibcode:
- 1982PNAS...79.7944D