Fluorescence Polarization (FP) assays are a powerful analytical technique widely utilized in the life sciences. This method measures changes in the polarization of light emitted by fluorescent molecules, offering a sensitive way to study molecular interactions in solution. The technique is valuable for its ability to provide quantitative analysis of diverse molecular interactions and enzyme activities, making it a routine tool in various research and clinical settings.
Understanding Fluorescence Polarization
The scientific principle behind fluorescence polarization centers on how fluorescent molecules behave when illuminated by polarized light. When a fluorescent molecule absorbs light, it enters an excited state, and upon returning to its ground state, it emits light. If the excitation light is polarized, the emitted light will also be polarized to a certain degree.
The degree of polarization in the emitted light is inversely related to the speed at which the fluorescent molecule rotates during its excited lifetime. Small molecules tumble rapidly in solution, causing the emitted light to become largely depolarized. Conversely, if a small fluorescent molecule binds to a larger molecule, its rotational motion slows considerably. This slower rotation means the emitted light retains a higher degree of polarization, remaining closer to the plane of the excitation light. This change in the observed polarization, from low to high or vice versa, provides a direct measure of molecular binding or dissociation events. This rotational movement, often referred to as Brownian motion, directly impacts the extent of light depolarization.
The Mechanics of an FP Assay
An FP assay detects changes in molecular size due to binding events. It involves a fluorescently labeled molecule, or “tracer,” and a potential binding partner in a sample. The tracer is typically a small molecule, peptide, or protein, generally less than 10 kilodaltons (kD) in size, that can be covalently linked to a fluorophore.
The assay begins by mixing the fluorescently labeled tracer with the sample containing the binding partner. If the tracer binds to the larger partner, its effective molecular size increases, slowing its rotational speed. The mixture is then excited with plane-polarized light, and the polarization of the emitted fluorescence is measured. A specialized instrument, typically a fluorescence plate reader equipped with polarizers for both excitation and emission, detects the fluorescence intensity in planes parallel and perpendicular to the excitation light. A change in polarization, expressed in milliP (mP) units, indicates that the small tracer has bound to a larger molecule, resulting in slower rotation and more polarized emitted light.
Key Applications of FP Assays
FP assays are widely used across various scientific and medical fields due to their homogeneous format, allowing for rapid and quantitative analysis without wash steps. In drug discovery, FP assays are employed for high-throughput screening of small molecule libraries to identify potential drug candidates. These assays can detect direct binding interactions between drug compounds and their target proteins, such as G protein-coupled receptors (GPCRs), kinases, phosphatases, and proteases.
FP assays are also valuable for studying protein-protein interactions (PPIs), protein-DNA interactions, and nucleic acid hybridization. For instance, a fluorescently labeled peptide representing a binding “hot spot” can show a high FP value when bound to its protein partner. If a small molecule inhibitor displaces this peptide, the FP value decreases due to the peptide’s increased rotational mobility. The homogeneous nature of FP assays, coupled with their non-radioactive detection and miniaturization capabilities, makes them highly suitable for screening thousands of compounds efficiently.
Beyond drug discovery, FP assays have found applications in clinical diagnostics for detecting specific molecules in patient samples, such as hormones, toxins, or therapeutic drug levels. Fluorescence Polarization Immunoassays (FPIA) are a class of FP assays that use a fluorophore-bound antigen which, when bound to an antibody, increases fluorescence polarization. This change is proportional to the amount of antigen in the sample, allowing for quantification. FP is also used in general research to investigate enzyme activities by monitoring the change in molecular volume upon substrate binding or product formation.
Factors Influencing FP Assay Results
Several parameters can influence the accuracy and reliability of Fluorescence Polarization assay results. The size difference between the fluorescently labeled molecule (tracer) and its binding partner is a significant factor. A substantial change in molecular volume upon binding, ideally a factor of five or more, yields a more robust and detectable polarization change. For example, a small tracer of around 2 kilodaltons (kD) binding to a protein of 10 kD or larger would provide a good signal.
The choice of fluorophore is also important; while FP is generally less dye-dependent than fluorescence intensity assays, the fluorophore’s excited-state lifetime influences the assay’s dynamic range. Stable fluorophores that maintain their fluorescent properties throughout the experiment contribute to more robust measurements. Temperature and solvent viscosity directly impact molecular rotation; an increase in temperature accelerates Brownian motion, decreasing polarization, while higher viscosity slows it down, increasing polarization. Maintaining a constant temperature and consistent buffer viscosity is important for accurate results. The purity of the tracer and binding partner, along with the absence of contaminants that scatter light or exhibit background fluorescence, are also important considerations for assay quality.