Calcium binding to fluorescent calcium indicators: calcium green, calcium orange and calcium crimson.

The recently introduced fluorescent calcium sensitive indicators calcium green, calcium orange and calcium crimson suggest important improvements and advantages to detect small calcium transients at low indicator concentrations. Thermodynamic dissociation constants and dissociation rate constants of calcium green, calcium orange and calcium crimson were measured by use of fluorescence titration and stopped flow fluorescence, respectively. Calcium binding to the indicators conforms to a 1:1 calcium:indicator complex although at high concentrations of calcium the fluorescence properties deviate somewhat from the behaviour predicted by the simple model. Dissociation of the calcium-indicator complex was found to be monoexponential under all conditions examined. The affinity for calcium of the three indicators generally increases with raising temperatures (Kd at 11.5 degrees C and 39.7 degrees C (nM): 261, 180 for calcium green; 527, 323 for calcium orange; 261, 204 for calcium crimson) and pH (Kd at pH 6.42 and 7.40 (nM): 314, 226 for calcium green; 562, 457 for calcium orange; 571, 269 for calcium crimson). The changes of the thermodynamic dissociation constant are mainly caused by changes of the association rate constant. The temperature dependence of calcium binding to the indicators revealed that this process is entropically favoured at ambient temperature.