Meuwis K, Boens N, De Schryver F C, Gallay J, Vincent M
Department of Chemistry, Katholieke Universiteit Leuven, Heverlee, Belgium.
Biophys J. 1995 Jun;68(6):2469-73. doi: 10.1016/S0006-3495(95)80428-5.
The fluorescent indicator PBFI is widely used for the determination of intracellular concentrations of K+. To investigate the binding reaction of K+ to PBFI in the ground and excited states, steady-state and time-resolved measurements were performed. The fluorescence decay surface was analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution at pH 7.2: k01 = 1.1 x 10(9) s-1, k21 = 2.7 x 10(8) M-1s-1, k02 = 1.8 x 10(9) s-1, and k12 = 1.4 x 10(9) s-1. k01 and k02 denote the respective deactivation rate constants of the K+ free and bound forms of PBFI in the excited state. k21 represents the second-order rate constant of binding of K+ to the indicator in the excited state whereas k12 is the first-order rate constant of dissociation of the excited K(+)-PBFI complex. From the estimated values of k12 and k21, the dissociation constant Kd* in the excited state was calculated. It was found that pKd* (-0.7) is smaller than pKd (2.2). The effect of the excited-state reaction can be neglected in the determination of Kd and/or the K+ concentration. Therefore, intracellular K+ concentrations can be accurately determined from fluorimetric measurements by using PBFI as K+ indicator.
荧光指示剂PBFI被广泛用于测定细胞内K+的浓度。为了研究K+与PBFI在基态和激发态下的结合反应，进行了稳态和时间分辨测量。用全局房室分析对荧光衰减表面进行分析，得出在pH 7.2的水溶液中室温下速率常数的以下值：k01 = 1.1×10(9) s-1，k21 = 2.7×10(8) M-1s-1，k02 = 1.8×10(9) s-1，以及k12 = 1.4×10(9) s-1。k01和k02分别表示激发态下PBFI的K+游离和结合形式的失活速率常数。k21代表K+与激发态指示剂结合的二级速率常数，而k12是激发态K(+)-PBFI络合物解离的一级速率常数。根据k12和k21的估计值，计算了激发态下的解离常数Kd*。发现pKd*（-0.7）小于pKd（2.2）。在测定Kd和/或K+浓度时，激发态反应的影响可以忽略不计。因此，通过使用PBFI作为K+指示剂，可以通过荧光测量准确测定细胞内K+浓度。
