A micro-spectrophotometric study on the spectral properties of phenol red injected into cytoplasm for pH_i determination

The spectral properties of phenol red, used to determine intracellular pH (pH_i), have been studied in the myoplasm of cultured rat skeletal muscle cells, by means of a micro-spectrophotometric technique. Two sets of intracellular calibration spectra at different dye concentrations were established in the presence of nigericin. Intracellular spectra of phenol red differ from the absorption spectra obtained in buffered solutions in the following ways: (1) Considerably greater absorbance in the violet region, owing to scattering of light by intracellular particles, which can be corrected by applying an energy transfer model derived from the work of Kubelka and Munk (Z Tech. Phys. 12 (1931) 593). (2) Slight spectral red-shift and an increase in the dye pK_d, assumed to be due to interaction of the dye with intracellular constituents, especially soluble proteins, since the same spectral modifications are observed in buffered solutions containing bovine serum albumin. The potential advantages of using phenol red to determine pH_i are the simple dye-H⁺ interaction, both in buffered solutions and in myoplasm, and the absence of dye selfassociations at the high dye concentrations required to perform intracellular measurements. Finally, our results show that absolute pH_i can be measured to an accuracy of ±0.02 pH units using phenol red, if complete absorption spectra and intracellular spectral calibrations are available.