Kowalczyk A, Boens N, Meuwis K, Ameloot M
Institute of Physics, Nicholas Copernicus University, Torun, Poland.
Anal Biochem. 1997 Feb 1;245(1):28-37. doi: 10.1006/abio.1996.9932.
A test based on time-resolved fluorescence experiments is proposed to assess the interference of an excited-state reaction with the fluorimetric determination of the ground-state dissociation constant Kd of ion [symbol: see text] fluorescent indicator complexes. If an inflection point occurs in the plot of the fluorescence signal vs -log [ion] in the concentration range of the ion where both decay times are invariant, this inflection point can be associated with the correct Kd. In contrast, the inflection point(s) in the concentration range where the decay times vary cannot be attributed to Kd. The test is applied to the fluorescent ion indicators SBFI (for Na+), PBFI (for K+), and Fura-2 (for Ca2+). In all three cases the decay times are invariant in the physiological concentration ranges of the respective ions, indicating that the fluorimetrically determined Kd values are actually the true Kd values.
本文提出了一种基于时间分辨荧光实验的测试方法，用于评估激发态反应对离子[符号：见原文]荧光指示剂配合物基态解离常数Kd荧光测定的干扰。如果在两种衰减时间均不变的离子浓度范围内，荧光信号与-log[离子]的关系图中出现拐点，则该拐点可与正确的Kd相关联。相反，在衰减时间变化的浓度范围内的拐点不能归因于Kd。该测试应用于荧光离子指示剂SBFI（用于Na+）、PBFI（用于K+）和Fura-2（用于Ca2+）。在所有三种情况下，衰减时间在各自离子的生理浓度范围内是不变的，这表明荧光测定的Kd值实际上是真实的Kd值。
