Van den Bergh V, Boens N, De Schryver F C, Ameloot M, Steels P, Gallay J, Vincent M, Kowalczyk A
Department of Chemistry, Katholieke Universiteit Leuven, Heverlee, Belgium.
Biophys J. 1995 Mar;68(3):1110-9. doi: 10.1016/S0006-3495(95)80285-7.
The photophysics of the complex forming reaction of Ca2+ and Fura-2 are 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 BAPTA as Ca2+ buffer: k01 = 1.2 x 10(9)s-1, k21 = 1.0 x 10(11) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 2.2 x 10(7) s-1, and with EGTA as Ca2+ buffer: k01 = 1.4 x 10(9) s-1, k21 = 5.0 x 10(10) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 3.2 x 10(7) s-1. k01 and k02 denote the respective deactivation rate constants of the Ca2+ free and bound forms of Fura-2 in the excited state. k21 represents the second-order rate constant of binding of Ca2+ and Fura-2 in the excited state, whereas k12 is the first-order rate constant of dissociation of the excited Ca2+:Fura-2 complex. The ionic strength of the solution was shown not to influence the recovered values of the rate constants. From the estimated values of k12 and k21, the dissociation constant Kd in the excited state was calculated. It was found that in EGTA Ca2+ buffer pKd (3.2) is smaller than pKd (6.9) and that there is negligible interference of the excited-state reaction with the determination of Kd and [Ca2+] from fluorimetric titration curves. Hence, Fura-2 can be safely used as an Ca2+ indicator. From the obtained fluorescence decay parameters and the steady-state excitation spectra, the species-associated excitation spectra of the Ca2+ free and bound forms of Fura-2 were calculated at intermediate Ca2+ concentrations.
利用稳态和时间分辨荧光测量研究了Ca2+与Fura-2形成复合物反应的光物理过程。采用全局房室分析对荧光衰减曲线进行分析，得出在以BAPTA作为Ca2+缓冲剂的室温水溶液中速率常数的以下值：k01 = 1.2×10(9)s-1，k21 = 1.0×10(11) M-1 s-1，k02 = 5.5×10(8) s-1，k12 = 2.2×10(7) s-1；在以EGTA作为Ca2+缓冲剂的情况下：k01 = 1.4×10(9) s-1，k21 = 5.0×10(10) M-1 s-1，k02 = 5.5×10(8) s-1，k12 = 3.2×10(7) s-1。k01和k02分别表示激发态下Fura-2的游离Ca2+形式和结合Ca2+形式的失活速率常数。k21代表激发态下Ca2+与Fura-2结合的二级速率常数，而k12是激发态Ca2+:Fura-2复合物解离的一级速率常数。结果表明，溶液的离子强度不影响速率常数的恢复值。根据k12和k21的估计值，计算了激发态下的解离常数Kd。发现在EGTA Ca2+缓冲剂中pKd（3.2）小于pKd（6.9），并且激发态反应对通过荧光滴定曲线测定Kd和[Ca2+]的干扰可忽略不计。因此，Fura-2可安全用作Ca2+指示剂。根据获得的荧光衰减参数和稳态激发光谱，计算了中间Ca2+浓度下Fura-2的游离Ca2+形式和结合Ca2+形式的物种相关激发光谱。
