The kinetics of Ca2+ dissociation from fura-2 and indo-1 were measured using a stopped-flow spectrofluorimeter. The dissociation rate constants were 84 s−1 and 130 s−1, respectively, in 0.1 M KCl at 20°C. The rate constants were insensitive to pH over the range 7.0 to 8.0. The second order association rate constants were estimated indirectly to be in the region of 5 × 108 M−1·s−1 and thus approach the diffusion-controlled limit. The results demonstrate that these new generation indicators are well-suited to measure rapid changes in concentration of intracellular Ca2+.
