Non-hyperbolic calcium calibration curve of Fura-2: implications for the reliability of quantitative Ca2+ measurements

The fluorescence probe Fura-2 is widely applied for the quantitative determination of cellular free Ca2+ concentration. Generally, a hyperbolic calibration curve has been taken as a basis. The in vitro calibration of Fura-2 performed with free Ca2+ concentrations ranging from 10 nM to about 9 mM demonstrates a non-hyperbolic curve. Assuming two Ca2+ binding sites of Fura-2 dissociation constants of 190 nM and 176 microM were estimated. The analysis of the calibration data by the equation introduced by Grynkiewicz et al. [Grynkiewicz G., Poenie M., Tsien R.Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440-3450] shows that the inclusion of Ca2+ concentrations in the range of the low-affinity binding site leads to an overestimation of the dissociation constant normally used within this method. The Rmax value conventionally estimated at a nominal saturating free Ca2+ concentration increases if concentrations in the range of the low-affinity binding site are selected for this purpose. Provided that millimolar free Ca2+ concentrations are applied for the determination of Rmax, the experimentally-estimated free Ca2+ concentration should be considerably lower than the real cellular concentration. This bias is especially to be expected if free Ca2+ concentrations exceeding the high-affinity Ca2+ binding site of Fura-2 are measured. With the use of approximately 10 microM free Ca2+ for the determination of Rmax the contribution of the low-affinity binding site is negligible. Data for cellular free Ca2+ calculated on the basis of Rmax values estimated with Ca2+ concentrations in the millimolar range should be considered with caution.