Van Craenenbroeck E, Engelborghs Y
Laboratory of Biomolecular Dynamics, University of Leuven, Heverlee, Belgium.
Biochemistry. 1999 Apr 20;38(16):5082-8. doi: 10.1021/bi9821925.
Fluorescence correlation spectroscopy (FCS) is a new technique that allows the determination of the diffusion constant of a fluorescent molecule in solution. Also, the binding of the fluorescent molecule to a target can be analyzed, if the difference in the diffusion coefficients of the free and bound ligand is sufficiently large. With FCS, the interaction between fluorescein-colchicine (FC) and tubulin has been studied in vitro. A fast and reversible binding is observed with an association constant at room temperature of (3.9 +/- 0.1) x 10(4) M-1. No competition with colchicine is seen, indicating that FCS reveals the existence of a new binding site on tubulin. FCS is not able to show the binding of FC to the original colchicine binding site, even though it exists, because the fluorescence of FC is strongly quenched upon binding to this site. This quenching is evident in spectrofluorometry experiments, revealing a slow binding of FC to tubulin that is subject to competition with colchicine. FCS allows the determination of the diffusion coefficients of both free and bound fluorescent colchicine which were found to be (2.6 +/- 0.2) x 10(-)10 and (2.0 +/- 0.2) x 10(-)11 m2 s-1, respectively. It can be concluded that fluorescent labeling, especially of small molecules, can interfere considerably with the binding behavior that is being studied. Although general qualitative effects in vivo are similar for colchicine and its fluorescein derivative, this quantitative study of the binding to tubulin presents a nuanced view, and the existence of a second binding site for FC can even explain some conflicting indications in the literature.
荧光相关光谱法（FCS）是一种新技术，可用于测定荧光分子在溶液中的扩散常数。此外，如果游离配体和结合配体的扩散系数差异足够大，还可以分析荧光分子与靶标的结合情况。利用FCS，已在体外研究了荧光素 - 秋水仙碱（FC）与微管蛋白之间的相互作用。观察到一种快速且可逆的结合，室温下的缔合常数为（3.9±0.1）×10⁴ M⁻¹。未观察到与秋水仙碱的竞争，这表明FCS揭示了微管蛋白上一个新结合位点的存在。即使存在原始的秋水仙碱结合位点，FCS也无法显示FC与该位点的结合，因为FC与该位点结合后荧光会强烈猝灭。这种猝灭在荧光光谱实验中很明显，表明FC与微管蛋白的结合缓慢且会受到秋水仙碱的竞争。FCS可以测定游离和结合的荧光秋水仙碱的扩散系数，分别为（2.6±0.2）×10⁻¹⁰和（2.0±0.2）×10⁻¹¹ m² s⁻¹。可以得出结论，荧光标记，尤其是小分子的荧光标记，可能会对正在研究的结合行为产生相当大的干扰。尽管秋水仙碱及其荧光素衍生物在体内的一般定性作用相似，但对与微管蛋白结合的这种定量研究呈现出细微差别，并且FC第二个结合位点的存在甚至可以解释文献中的一些相互矛盾的迹象。
