Continuous measurement of mitochondrial pH gradients in isolated hepatocytes by difference ratio spectroscopy

Philip J. Thomas, Larry D. Gaspers, Christine Pharr, John A. Thomas

Research output: Contribution to journalArticlepeer-review

15 Scopus citations


The pH gradient, ΔpH, present across the inner mitochondrial membrane in isolated rat hepatocytes was continuously monitored with a novel spectroscopic technique that utilizes the weak acid fluorescein. Unlike most cytosolic pH indicators, such as 2′,7′-bis(carboxyethyl)-5, (6)-carboxyfluorescein (BCECF), fluorescein freely distributes between the cytosolic and mitochondrial compartments. As is typical for weak acids, the distribution between these two compartments is governed by the magnitude of the pH gradient. Since fluorescein has two ionizable groups, the fluorescein dianion is concentrated in the mitochondrial compartment 100-fold per ΔpH unit. In this compartment, fluorescein absorbance (or excitation) spectra are red-shifted about 6-8 nm in the matrix environment, as compared to the cytosolic dye at equivalent pH values. The combination of favorable mitochondrial accumulation and red-shifted spectra enables mitochondrial pH to be continuously monitored qualitatively in whole cells by dual wavelength spectroscopy (510 minus 540 nm). When the cytosolic pH is determined by independent means, the mitochondrial pH can be quantitated, based on the theoretical dependence of the fluorescein distribution ratio on ΔpH, the ratio of cytosolic to mitochondrial volumes, and the known extinction coefficients for the dye in the cytosolic and mitochondrial compartments. The sensitivity of the method for following kinetic responses in mitochondrial pH is especially noteworthy; a 0.1-unit change in ΔpH is easily distinguished, with a time resolution of less than a second.

Original languageEnglish (US)
Pages (from-to)250-260
Number of pages11
JournalArchives of Biochemistry and Biophysics
Issue number1
StatePublished - Jul 1991
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology


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