PHOTOPHYSICS OF THE FLUORESCENT Ca2+ INDICATOR QUIN-2 |
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Authors: | Viviane Van den Bergh Noël Boens Frans C De Schryver Jacques Gallay Michel Vincent |
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Institution: | Department of Chemistry, Katholieke Universiteit Leuven, B-3001 Heverlee, Belgium;Laboratoire pour l'Utilisation du Rayonnement Electromagnétique, Centre Universitaire Paris-Sud, 91405 Orsay, France |
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Abstract: | The photophysics of the complex forming reaction between Quin-2 and Ca2+ were investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with EGTA as Ca2+ buffer: k01= 8.6 times 108 s?1, k21= 1 times 1011M?1 s?1, k02= 8.8 times 107 s?1, k12= 4 times 104 s?1. k01 and k02 denote the respective deactivation rate constants of the Ca2+ free and bound forms of Quin-2 in the excited state. The constant k21 represents the second-order rate constant of binding of Ca2+ and Quin-2 in the excited state while k12 is the first-order rate constant of dissociation of the excited Ca2+:Quin-2 complex. From the estimated values of k12 and k21 the dissociation constant Kd* in the excited state was calculated. It was found that pKd* (6.4) is slightly smaller than pKd (7.2). There was no interference of the excited-state complex forming reaction with the determination of Kd. Intracellular Ca2+ concentrations can thus accurately be determined from fluorometric measurements using Quin-2 as Ca2+ indicator. |
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