通过全内反射荧光光漂白恢复技术测量抗二硝基苯基单克隆Fab在支持的磷脂单分子层上的结合动力学。
Binding kinetics of an anti-dinitrophenyl monoclonal Fab on supported phospholipid monolayers measured by total internal reflection with fluorescence photobleaching recovery.
作者信息
Pisarchick M L, Gesty D, Thompson N L
机构信息
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
出版信息
Biophys J. 1992 Jul;63(1):215-23. doi: 10.1016/S0006-3495(92)81592-8.
Fluorescence photobleaching recovery with total internal reflection illumination (TIR-FPR) has been used to measure the dissociation kinetics of a fluorescein-labeled anti-dinitrophenyl monoclonal Fab specifically bound to supported monolayers composed of a mixture of dipalmitoylphosphatidylcholine and dinitrophenyl-conjugated dipalmitoylphosphatidylethanolamine. The fluorescence recovery curves were not monoexponential; when analyzed as a sum of two exponentials, the rates and fractional recoveries were approximately 1 s-1 (approximately 50%) and approximately 0.1 s-1 (approximately 30%). The data did not change as a function of the Fab solution concentration, indicating that the fluorescence recovery curves were not influenced by the rate of diffusion in bulk solution. Also, the recovery curves were independent of the size of the illuminated area, indicating that surface diffusion did not significantly contribute to the rate and shape of the fluorescence recovery. The measured off rates and apparent association constant (1.6 x 10(5) M-1) were analyzed with the theoretical formalism for a proposed mechanism that accounts for the nonmonoexponential kinetics.
采用全内反射照明的荧光光漂白恢复技术（TIR - FPR）来测量与由二棕榈酰磷脂酰胆碱和二硝基苯基共轭二棕榈酰磷脂酰乙醇胺混合物组成的支持单层特异性结合的荧光素标记抗二硝基苯基单克隆Fab的解离动力学。荧光恢复曲线不是单指数的；当作为两个指数的和进行分析时，速率和分数恢复率分别约为1 s⁻¹（约50%）和约0.1 s⁻¹（约30%）。数据不会随Fab溶液浓度而变化，这表明荧光恢复曲线不受本体溶液中扩散速率的影响。此外，恢复曲线与照明区域的大小无关，这表明表面扩散对荧光恢复的速率和形状没有显著贡献。利用一种解释非单指数动力学的提议机制的理论形式对测得的解离速率和表观缔合常数（1.6×10⁵ M⁻¹）进行了分析。
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