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Fluorescence polarization (FP) is a useful analytical technique that can measure the extent to which a fluorescent tracer (typically a small molecule) binds to a large molecule, such as an enzyme, nucleic acid, or antibody. Since 1980, FP immunoassays have been used extensively in the clinical laboratory to measure the concentration of drugs, both therapeutic and illicit, in biological fluids. Recently, LJL Biosystems, PanVera, and other companies have adapted the FP technique to the HTS environment.

FP theory states that when a molecule is excited with plane-polarized light of the correct wavelength, it will fluoresce in the same plane after its characteristic emission lifetime, which is typically a few nanoseconds. During this time, the molecule will have tumbled randomly with respect to the original plane of excitation. If the molecule tumbles quickly with respect to the fluorescence lifetime, the fluorescence will be depolarized. However, if the molecule tumbles slowly with respect to the fluorescence lifetime, the observed fluorescence will remain significantly polarized (Figure 2).

In general, a molecule's rate of tumbling (more formally known as the rotational relaxation time) is directly proportional to its molecular volume at constant temperature and viscosity. Small molecules tumble rapidly, large molecules tumble slowly. If a small fluorescent molecule is bound to a large molecule, however, then it will tumble slowly.

By measuring the extent of fluorescence polarization, this method can determine binding equilibrium and the competition for binding at a site. For example, suppose a company wants to screen for compounds that are enzyme inhibitors and that bind strongly to an active site. The FP assay would use a fluorescent marker that also binds to that enzyme's active site. Stronger inhibitors would compete more successfully for the binding sites, thus releasing more of the fluorescent marker into the unbound state. These samples would show a greater degree of random depolarization.

The FP technique has several advantages. It can be done easily in homogeneous form. It serves as a direct and fairly universal probe for binding. And, because FP measures the ratio of two intensities (the bound versus unbound marker), each sample has an internal calibration. Therefore, several experimental limitations are minimized, including variations in intensity because of source fluctuations, fluorescence quenching by other species in solution that absorb at either excitation or emission wavelengths, and scattering caused by turbidity. FP is particularly well suited for assays that rely on small ligands binding to large biomolecules, including kinase assays, protease assays, and immunoassays.

Nevertheless, the FP technique does have its limitations. This method does not increase the sensitivity of the measurement with respect to background, so sensitivity questions must be looked at carefully. In cases that require highly sensitive assays because of the limited availability of targets (such as membrane receptors), FP assay techniques have not been the best choice in the past. Nonetheless, new technologies, such as high-efficiency FP (HEFP, LJL Biosystems), are expanding the sensitivity range of assays and have been used successfully with receptor binding assays.

Another consideration is that the method requires a ligand or tracer that is fluorescent and can bind to the target site. In some cases, appropriate tracers are already available commercially. LJL's Sportsman points out, "If a fluorescent-labeled peptide or small molecule is already available for the target that you are interested in, then you can very quickly put together a polarization assay." In other cases, however, appropriate tracers must be developed either by the pharmaceutical company or by a company that specializes in assay development (LJL Biosystems and PanVera are two such companies). This can be challenging in those cases that involve screening against a target in which the native ligands are small organic molecules such as nonpeptide neurotransmitters.