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

The kinetics of the Quin 2-Ca2+ interaction have been studied using stopped-flow fluorimetry. Mixing the Quin 2-Ca2+ complex with a large excess of EGTA, EDTA or MgCl2 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 degrees C, +/- 3mM Mg2+. 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 greater than or equal to 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 Ca2+ transients.