The application of fluorescent polarization/anisotropy is based on the fact that molecules will rotate more slowly in solution when bound to a large complex, due to hydrodynamic effects. A fluorescently-labelled molecule will have a high fluorescence polarization/anisotropy when bound to a macromolecule complex than when alone, as the fluorophore is less mobile. Therefore, this method can be applied to measure complex formation (protein:protein, protein:DNA and protein:ATP). This is particularly useful because fluorescence intensity changes do not always occur and sometimes numerous fluorescent phases occur, which makes it difficult to distinguish which phase corresponds to binding or to conformation changes. Fluorescence polarization/anisotropy circumvents this problem by directly reporting upon complex formation.
Fluorescence anisotropy uses polarized light to selectively excite fluorophores whose absorption transition dipole is parallel to that of the polarized excited light. This process is termed ‘photoselection’. The range of dipoles selected is dependent upon the angle of the dipole in relation to the excitation light. The chance of a dipole absorbing a photon is proportional to the cosine squared of the angle between the excitation light and the dipole. Following photoselection; fluorophores can tumble in solution during the excited state and emit light in an alternative plane to that of the excited light plane. When a fluorophore excited by polarised light is immobile during its fluorescence lifetime (typically 0.1 – 30 nanoseconds), the polarisation of the emitted light will also be unchanged. However, if the fluorophore is mobile during this time, the orientation of the molecules will be randomised and the emitted light will be less polarised.
Fluorescence Anisotropy. (A) Polarised light is selected using calcite prisms which separate vertical and horizontal polarised light. Vertically polarised light then excites the fluorophores with the excitation transition dipoles, as shown by the line in the diagram, parallel to the excitation plane (bright blue), in a process called ‘photoselection’. (B) A sample containing a fluorescently labelled sample is excited with vertically polarized light. Fluorophores in the excited state are able to tumble in solution within the fluorescence lifetime. The rate of tumbling is related to the size of the macromolecular complex. A large complex is depicted by the protein shown in green. Emission is measured through polarizers that are parallel and perpendicular to the plane of the excitation.
The fluorescence lifetime is important for these measurements. If the lifetime is too short, the molecule will not have the time to rotate, so the emitted light will always appear polarised,. However, if the lifetime is too long, the emitted light will always be depolarised. For many biological systems, the fluorescence lifetime should be relatively short. However, if the fluorescently labelled species are very large, then a longer lifetime is required to allow anisotropy measurements. For instance, fluorescein is a suitable fluorophore for fluorescence anisotropy measurements, as its lifetime (4 ns) is long enough for many biological applications.
One of the first equations to describe polarisation (P) was proposed by Perrin is 1926:
Where P0 is the limiting polarisation of the fluorophore, typically 0.5, r is the Debye rotational relaxation time which is dependent upon the volume and shape (molecular weight) of the rotating species, and t is the fluorescence lifetime. The Equation above is simplified for anisotropy (r) here:
Where r0 is the limiting anisotropy of the fluorophore in solution, typically 0.4, and tc is the rotational correlation time (r = 3tc). Therefore, larger molecules or complexes have a slower rotation correlation time in relation to the fluorescence lifetime, and thus a higher anisotropy.
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