PHOTOPHYSICS OF THE FLUORESCENT CA2-2( INDICATOR QUIN)

The photophysics of the complex forming reaction between Quin-2 and Ca 2+ 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 con stants at room temperature in aqueous solution with EGTA as Ca2+ buffe r: k(01) = 8.6 x 10(8) s(-1) k(21) = 1 x 10(11) M(-1) s(-1), k(02) = 8 .8 x 10(7) s(-1), k(12) = 4 x 10(4) s(-1). k(01) and k(02), denote the respective deactivation rate constants of the Ca2+ free and bound for ms of Quin-2 in the excited state. The constant k(21) represents the s econd-order rate constant of binding of Ca2+ and Quin-2 in the excited state while k(12) is the first-order rate constant of dissociation of the excited Ca2+:Quin-2 complex. From the estimated values of k(12) a nd k(21) the dissociation constant K-d in the excited state was calcu lated. It was found that pK(d) (6.4) is slightly smaller than pK(d) ( 7.2). There was no interference of the excited-state complex forming r eaction with the determination of K-d. Intracellular Ca2+ concentratio ns can thus accurately be determined from fluorometric measurements us ing Quin-2 as Ca2+ indicator.