Introduction to Anisotropy

Fluorescence polarization (FP) as an analysis technique is based on the finding that emission from a fluorophore excited by plane polarized light is depolarized by rotational diffusion of the molecule during the emission lifetime of the fluorophore. This theory was first described by Francis Perrin in 1926 [34]. FP can be used to determine shifts in molecular mass, as mass changes will alter the molecular rotation of the labeled molecule and therefore change the intensity of the FP readout in either the parallel or perpendicular channel. Below is the equation for FP denoted P for polarization. The equation for anisotropy is similar, with the only exception being that the intensity (I) horizontal in the denominator is multiplied by a factor of two.

Therefore, the terms FP and fluoresce anisotropy (FA) can be used interchangeably, although

FIGURE 1

It can be clearly seen in the above equation that concentration is not a factor as long as the fluorescent signal is within the limits of detection for the experimental instrument[35]. In

general, a greater anisotropy signal is due to more light being emitted in the parallel intensity and therefore slower diffusion, which can then be related to a relatively larger hydrodynamic radius.

This is useful in determining protein protein interactions (PPI) and their inhibitors in a high throughput manner. When associated, protein -protein complexes will be larger and therefore rotate slower than when the complex is disrupted by the addition of a molecule. Upon

disruption, the labeled species is free of its partner and will therefore be smaller and rotate faster, emitting more light in the perpendicular plane leading to a reduced anisotropy value [36], [37].

This ability to deduce the relative change in hydrodynamic radius, and the concentration independence of the readout, are at the heart of the following chapters, as we build a case for utilizing FA in determining and quantifying the interfacial forces of the protein-detergent complex.

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