Kinetics of calcium binding to fluo-3 determined by stopped-flow fluorescence

Abstract

The kinetics of Ca 2+ dissociation from fluo-3 was measured using stopped flow fluorimetry. Analysis of dissociation revealed, in contrast to other commonly used fluorescent Ca 2+ indicators, a biexponential behaviour with two distinct dissociation rates of 550 s −1 and 200 s −1 at physiological pH and room temperature. The dissociation rate constant of the fast phase increases to 700 s −1 at physiological temperature, whereas that of the slow phase does not change markedly. While the rate constants do not depend on pH between 6.6 and 7.8, the dissociation turns out to be monoexponential at pH 5.86. The association rate of Ca 2+ to fluo-3 could not be measured within the mixing dead time and is estimated to be above 10 9M −1 s −1. Since the rate constants of fluo-3 are larger than those of other fluorescent Ca 2+ indicators, fluo-3 is well suited for investigations of Ca 2+ oscillations in biological systems.