Fluorescein conjugates as indicators of subcellular pH. A critical evaluation

Fluorescein and tetramethylrhodamine conjugates to protein or dextran were used to determine subcellular pH. The pH dependence of fluorescence of fluorescein isothiocyanate (FITC) conjugates could be described by a single proton dissociation (pK'a approximately 6.8). This allowed pH to be derived accurately from spectra using the simple Henderson-Hasslebach equation. FITC and TRITC conjugates were delivered to mouse macrophage lysosomes by pinocytosis. Lysosomal pH was then determined in several different ways. First, by direct matching of the subcellular fluorescence spectrum with calibration spectra obtained in free solution. Secondly, monensin was used to equilibrate internal and extracellular pH. Subcellular pH could then be determined by the relative increase in fluorescence of the FITC conjugate without loss of probe from the lysosomes. This allowed the calibration of pH dependence with the probe in situ. Finally, macrophages were permitted to pinocytose FITC and TRITC dextran conjugates simultaneously. pH could be determined from the ratio of emissions from the two dyes within the lysosomes. Each of these different methods gave a similar value for lysosomal pH (4.8 +/- 0.1).