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note
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In cases where a relatively small fluorescent probe is attached to a larger macromolecule, the observed steady-state fluorescence anisotropy from the probe results from a combination of local probe motion, segmental motion of a subregion of the entire macromolecule and the probe, and/or the global motion of the macromolecule. As a result, if there is local/segmental dynamics, which is likely here, the measured experimental steady-state fluorescence anisotropy is biased toward lower values in comparison to what the value would be if the probe did not undergo local/segmental motion. Thus, the steady-state anisotropy measurements tend to underestimate intermolecular association if the associating species are flexible, and the probe can undergo motion independent of the macromolecule.
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