Time-Dependent Fluorescence Spectroscopy to Quantify Complex Binding Interactions

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.