Stopped-flow kinetics of the interaction of the fluorescent calcium indicator Quin 2 with calcium ions

Abstract

The kinetics of the Quin 2-Ca 2+ interaction have been studied using stopped-flow fluorimetry. Mixing the Quin 2-Ca 2+ complex with a large excess of EGTA, EDTA or MgCl 2 resulted in first order dissociation kinetics. The observed dissociation rate increased slightly with increasing EGTA concentration yielding a limiting value of 83±4 s −1 for the dissociation rate constant (k −) at pH 7.2, 37°C, ± 3mM Mg 2+. The temperature dependence of the dissociation was weak (activation energy = 22±1 kJ/mol) and around neutral pH the pH dependence was negligible. The association reaction was too fast to be monitored directly. From this and the instrument dead-time, the second order rate constant k + was estimated to be ≥10 9 M −1s −1, in agreement with the calculation from k + = k − K . These data should be useful in evaluating the potential of Quin 2 to measure fast intracellular Ca 2+ transients.