ORGN 651 |
| The fluorescence of fluorescein bioconjugates can change depending on the local environment around the biomolecule. For example, fluorescein-labeled variants of ribonuclease A exhibit a modest fluorescence decrease upon binding the ribonuclease inhibitor protein (RI). This phenomenon can be used to measure the dissociation constant (Kd) of a RI•ribonuclease complex. The basis of this fluorescence modulation was determined to be a perturbation of the phenolic pKa (@ 6.4) of the fluorescein label. We hypothesized that “tuning” this pKa to more closely match the solution pH could increase the dynamic range of our assay. Accordingly, we synthesized and evaluated 2',7'-diethylfluorescein (DEF) derivatives which have higher phenolic pKa values. A DEF-ribonuclease conjugate provided a substantial improvement in assay performance, ultimately allowing miniaturization to a microplate format. This new assay facilitates the measurement of Kd values for RI•ribonuclease complexes and demonstrates that pKa-tuning of fluorescent labels can be a valuable assay optimization technique.
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Asymmetric Reactions, Combinatorial Chemistry, Molecular Recognition and Self-Assembly, Proteins, Peptides, Amino Acids and Enzyme Inhibitors
8:00 PM-10:00 PM, Wednesday, March 28, 2007 Hyatt Regency Chicago -- Riverside Center, Poster
Division of Organic Chemistry |
