Cheung A T, Johnson D A, Taylor P
Biophys J. 1984 Feb;45(2):447-54. doi: 10.1016/S0006-3495(84)84167-3.
We have studied the kinetics of N epsilon-fluorescein isothiocyanate-lysine-23 cobra alpha-toxin (FITC-toxin) binding to the membrane-associated acetylcholine receptor from the Torpedo californica electric organ. The fluorescent toxin not only enabled us to monitor the binding reaction continuously but also to examine simultaneously the enhancement of ligand fluorescence and the increase in steady state polarization of fluorescence associated with binding of the alpha-toxin. Over the range of concentrations employed, both parameters yielded identical kinetic constants, suggesting that the enhancement of fluorescence of fluorescein and its immobilization are occurring in the same time frame. Both an initial rate analysis and the integrated rate expression showed association to be a simple, reversible bimolecular process. The apparent second-order association rate constant derived from the integrated rate analysis was constant within a factor of 2 over a 40-fold concentration range (6.7 +/- 1.7 X 10(3) M-1 S-1). The unimolecular dissociation rate constant was found to be 3.3 +/- 0.5 X 10(-5) S-1.
我们研究了异硫氰酸荧光素 - 赖氨酸 - 23 眼镜蛇α - 毒素（FITC - 毒素）与加州电鳐电器官中膜结合型乙酰胆碱受体结合的动力学。这种荧光毒素不仅使我们能够连续监测结合反应，还能同时检测配体荧光的增强以及与α - 毒素结合相关的荧光稳态极化的增加。在所采用的浓度范围内，这两个参数得出了相同的动力学常数，表明荧光素荧光的增强及其固定化是在同一时间框架内发生的。初始速率分析和积分速率表达式均表明结合是一个简单的、可逆的双分子过程。通过积分速率分析得出的表观二级结合速率常数在 40 倍浓度范围内（6.7 ± 1.7×10³ M⁻¹ s⁻¹）的变化系数在 2 以内保持恒定。发现单分子解离速率常数为 3.3 ± 0.5×10⁻⁵ s⁻¹。
