Abstract
Measuring the binding affinity for proteins that can aggregate or undergo complex binding motifs presents a variety of challenges. In this study, fluorescence lifetime measurements using intrinsic tryptophan fluorescence were performed to address these challenges and to quantify the binding of a series of carbohydrates and carbohydrate-functionalized dendrimers to recombinant human galectin-3. Collectively, galectins represent an important target for study; in particular, galectin-3 plays a variety of roles in cancer biology. Galectin-3 binding dissociation constants (KD) were quantified: lactoside (73 ± 4 μM), methyllactoside (54 ± 10 μM), and lactoside-functionalized G(2), G(4), and G(6)-PAMAM dendrimers (120 ± 58 μM, 100 ± 45 μM, and 130 ± 25 μM, respectively). The chosen examples showcase the widespread utility of time-dependent fluorescence spectroscopy for determining binding constants, including interactions for which standard methods have significant limitations.
Highlights
Protein−ligand binding interactions are currently measured by many different methods.[1,2] Some of the most common methods include microcalorimetry, fluorescence intensity, absorbance intensity, fluorescence anisotropy, surface plasmon resonance, and the enzyme-linked immunosorbent assay (ELISA)
Binding studies with low-affinity monomeric ligands and proteins that are prone to oligomerization are readily obtainable even when only small changes in the fluorescence emission spectra occur upon ligand binding
Improved accuracy was achieved relative to steady-state fluorescence binding studies, and the fluorescence lifetime method of measuring binding constants can provide benchmark values for assessment of other techniques such as surface-based and steady-state studies
Summary
Protein−ligand binding interactions are currently measured by many different methods.[1,2] Some of the most common methods include microcalorimetry, fluorescence intensity, absorbance intensity, fluorescence anisotropy, surface plasmon resonance, and the enzyme-linked immunosorbent assay (ELISA). For G(4), the Hill coefficient, n, was determined to be 1.0 ± 0.2 which is consistent with noncooperative binding This deviation from the other glycodendrimers can be explained through the optimal flexibility and available surface area of G(4), as compared to G(2) and G(6), which allows for steric hindrance of lactoside dendrimer endgroups to play a less influential role in subsequent binding interactions.[51−54] Table 1 summarizes the reported binding and cooperativity constants. The limited sensitivity of the steady-state measurements and the corresponding large error bars at low concentrations of lactose likely cause the discrepancy in binding constants obtained using steady-state experiments as compared to values obtained using fluorescence lifetime measurements For both steadystate and lifetime protocols, control experiments were carried out to ensure that fluorescence was not altered by PBS additions, ruling out concentration effects, or by ligand additions independent of galectin-3, ruling out the introduction of unintended fluorophores
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