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Showing 1-2 of 2 resultsCells tightly regulate their contents. Still, nonspecific Coulombic interactions between cationic molecules and anionic membrane components can lead to adventitious endocytosis. Here, we characterize this process in a natural system. To do so, we create variants of human pancreatic ribonuclease (RNase 1) that differ in net molecular charge. By conjugating a small-molecule latent fluorophore to these variants and using flow cytometry, we are able to determine the kinetic mechanism for RNase 1 internalization into live human cells. We find that internalization increases with solution concentration and is not saturable. Internalization also increases with time to a steady-state level, which varies linearly with molecular charge. In contrast, the rate constant for internalization (t1/2 = 2 h) is independent of charge. We conclude that internalization involves an extracellular equilibrium complex between the cationic proteins and abundant anionic cell-surface molecules, followed by rate-limiting internalization. The enhanced internalization of more cationic variants of RNase 1 is, however, countered by their increased affinity for the cytosolic ribonuclease inhibitor protein, which is anionic. Thus, Coulombic forces mediate extracellular and intracellular equilibria in a dichotomous manner that both endangers cells and defends them from the potentially lethal enzymatic activity of ribonucleases.
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. 2ā,7ā-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 Zā-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.