pH-dependent intracellular quenching of the indicator carboxy-SNARF-1

Carboxy-SNARF-1 is an emission-changing, pH-sensitive probe for measurements of intracellular pH. However, the protonated and deprotonated forms of the dye interact differently with intracellular constituents, and this imposes new requirements on the calibration of the system. Whole spectra of intracellular and extracellular C.SNARF-1 were analyzed and showed (1) intracellular quenching which was significantly greater for the deprotonated form of the dye than for the protonated state and (2) a detectable change in pK _a. Importantly for avoiding damage to cells, this mathematical analysis allowed reference spectra for fully protonated and fully deprotonated dye to be obtained without a need for spectra measured at extreme values of pH. It is not known what constituent(s) of the intracellular milieu might be responsible for the changes in dye behavior in the cell. To address this question, preliminary experiments with cell-free buffers compared the pattern seen inside the cell with quenching by a protein (bovine serum albumin; BSA) or that due to ethanol. The BSA result was completely unlike the intracellular case in that the protonated form of the dye was quenched. Buffer containing ethanol, on the other hand, was able to mimic the essential features of the intracellular spectra.