Tuning the pKaof Fluorescein to Optimize Binding Assays

The phenolic pKa 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 pKa 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‘-Diethylfluorescein (DEF) has spectral properties similar to those of fluorescein but a higher phenolic pKa. Most importantly, free and RI-bound DEF−RNase A have pKa 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 Z‘-factor from −0.17 to 0.69. We propose that synthetic “tuning” of the pKa of fluorescein and other pH-sensitive fluorophores provides a general means to optimize binding assays.