Fluorescence Technologies for Monitoring Interactions Between Biological Molecules In Vitro - Progress in Molecular Biology and Translational Science - page 116

Small unbound molecule
(faster rotation)

Fluorescence
depolarized

Probe

(emitted light in a different
plane from excitation light)

Molecule

Polarized
excitation light

Fluorescence
polarized
(emitted light in the same
plane as excitation light)

Interacting
partner

Large complex molecule
(slower rotation)

Figure 4.5 Principle of fluorescence polarization. The interactions of a probe-associated
molecule with interacting partners enable the formation of a large complex which in-
duces polarization or increases the polarization value (fluorescence polarized), whereas
no polarization variations occur for the unbound state of the probe-associated molecule
(fluorescence depolarized).

is then “depolarized” ( Fig. 4.5 ).With regard to intermolecular interactions,
if a molecule is bound by a larger ligand, its effective molecular volume is
increased and its rotation is slowed so that the emitted light is in the same
plane as the plane-polarized light of excitation ( Fig. 4.5 ). In this case, bot-
h the bound and unbound states of the molecule have an intrinsic polariza-
tion value: a high (polarized) value for the bound state and a low
(depolarized) value for the unbound state. 17 Fluorescence polarization is
a weighted average of the two values, providing a direct evaluation of the
fraction of molecule/ligand binding. Thus fluorescence polarization mea-
surements are also indicative of the formation of larger molecule/ligand
complexes.
Fluorescence anisotropy is usually determined by the measurements of
fluorescence emission in parallel and perpendicular planes. The degree of
polarization ( p ) or anisotropy ( r ) is calculated according to the following
equations:

= and r =

p ¼ I k I ┬ I k þ I ┬ ¼ I k I ┬ I k þ 2 I ┬ ½ 4 : 2

where I k is the fluorescence emission measured in the plane parallel to the
plane of excitation and I ┬ is the fluorescence emission measured in the
plane perpendicular to the plane of excitation. Anisotropy and fluorescence
polarization approaches can be applied to study different biomolecular
interactions. 18