Non-calcium environmental sensitivity of intracellular Indo-1

Several mechanisms might account for the difference between spectra of Indo-1 measured on intracellular and on extracellular dye. Experiments to discriminate between various possibilities used quantitative analyses of intracellular and extracellular Indo-1 spectra. Intracellular calcium-free dye showed a 20-nm blue shift, when compared with the extracellular case. However, the calcium-bound spectrum was unaffected by the intracellular milieu. This eliminated the possibility that the spectral shift was due to calcium-independent unhydrolyzed dye. The spectral shift was reversible and was seen in resting cells as well as calcium-depleted cells. Since the apparent dissociation constant for calcium was not detectably different inside the cell, the possibility of competitive binding to another divalent cation, such as Zn²⁺, was eliminated as the principle source of the spectral shift. The shift appears to be due to a noncompetitive solvent effect on the emission of the calcium-free dye that is absent in the calcium-bound form.