The first application of fluorescence polarization to
monitor the binding of small
molecules to proteins was carried out by D. Laurence in 1952 using Gregorio Weber’s instrumentation in Cambridge. Specifically, Laurence studied the binding of numerous dyes, including
fluorescein, eosin, acridine and others, to bovine
serum albumin, and used the polarization data to estimate the binding constants.
Although many probes (such as fluorescein) do not
significantly alter their quantum
yield upon interaction with proteins, one should not take this fact for granted and would be well advised to check. If the quantum yield does in fact change, one can readily correct the fitting
equation to take the yield change
into account. In terms of anisotropy
the correct expression relating observed
anisotropy (r) to fraction of bound ligand (x), bound anisotropy (rb), free anisotropy (rf), and the quantum yield enhancement factor (g) is: