Tuning the pKa of fluorescein to optimize binding assays
Tuning the pKa of fluorescein to optimize binding assays
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DOI：
10.1021/ac070907g
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发表时间：
2007-09-01
影响因子：
7.4
通讯作者：
Raines, Ronald T.
中科院分区：
文献类型：
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作者：
Lavis, Luke D.;Rutkoski, Thomas J.;Raines, Ronald T.
荧光素的酚pK（a）随其环境而变化。染料的荧光也同样变化。因此，荧光的变化可以报告荧光素缀合物与另一分子的缔合。在这里，我们展示了如何用化学合成来优化这个过程。荧光素标记的模型蛋白，牛胰腺核糖核酸酶（RNase A）的荧光，结合其同源抑制蛋白（RI）后降低。游离和RI结合的荧光素-RNA酶A的pK（a）值分别为6.35和6.70，使得荧光素部分在生理pH下大部分未质子化，从而限制了测定的灵敏度。为了增加荧光素的pKa，因此，测定灵敏度，我们安装了一个电子给每个苯酚基团的邻位烷基。2 '，7'-二乙基荧光素（DEF）具有与荧光素相似的光谱性质，但具有更高的酚pKa。最重要的是，游离和RI结合的DEF-RNase A的pK（a）值分别为6.68和7.29，导致测定的灵敏度大幅增加。在pH 7.12的微孔板测定中使用DEF-RNase A而不是荧光素-RNase A将E因子从-0.17增加到0.69。我们建议，合成的“调谐”的荧光素和其他pH敏感的荧光团的pK（a）提供了一种通用的手段，以优化结合测定。
The phenolic pK(a) of fluorescein varies depending on its environment. The fluorescence of the dye varies likewise. Accordingly, a change in fluorescence can report on the association of a fluorescein conjugate to another molecule. Here, we demonstrate how to optimize this process with chemical synthesis. The fluorescence of fluorescein-labeled model protein, bovine pancreatic ribonuclease (RNase A), decreases upon binding to its cognate inhibitor protein (RI). Free and RI-bound fluorescein-RNase A have pK(a) values of 6.35 and 6.70, respectively, leaving the fluorescein moiety largely unprotonated at physiological pH and thus limiting the sensitivity of the assay. To increase the fluorescein pKa and, hence, the assay sensitivity, we installed an electron-donating alkyl group ortho to each phenol group. 2',7'-Diethyffluorescein (DEF) has spectral properties similar to those of fluorescein but a higher phenolic pKa. Most importantly, free and RI-bound DEF-RNase A have pK(a) values of 6.68 and 7.29, respectively, resulting in a substantial increase in the sensitivity of the assay. Using DEF-RNase A rather than fluorescein-RNase A in a microplate assay at pH 7.12 increased the E-factor from -0.17 to 0.69. We propose that synthetic "tuning" of the pK(a) of fluorescein and other pH-sensitive fluorophores provides a general means to optimize binding assays.