Investigation of Immunochemical Reactions by Fluorescence Polarization
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Fluorescence polarization provides the biologist and the chemist with a highly sensitive and versatile probe for investigating both the structural features and molecular motions of macromolecules. It provides information on the individual isolated molecule, as well as on the interactions occurring with other molecules, and is hence adaptable to a diversity of equilibrium and kinetic measurements over extremely wide ranges of concentration and time. The magnitude of the steady-state polarization (or the anisotropy) observed in the fluorescent light emitted from solutions is a function of several variables. It depends upon the relaxation time of the rotary Brownian motion, the decay time of the electronically excited state, and the relative orientations of the transition moments for absorption and emission. If the transient-state polarization is observed after the excitation has been cut off, then the polarization is a function of time as well. Measurements of both the steady-state and transient-state polarization are well known (Dandliker and deSaussure, 1970; Yguerabide, 1972) and are usually analyzed in terms of rotational motions of the fluorescent unit and of relaxation phenomena involving the macromolecule or solvent. The latter effects may be, in part, induced by the difference in free energy between the ground and the electronically excited states. In the last few years, there have been substantial advances in the theory of fluorescence polarization (Tao, 1969; Weber, 1971; Belford et al, 1972; Chuang and Eisenthal, 1972) together with improvements in instrumentation both for transient-state (Yguerabide, 1972) and for steady-state measurements (Kelly, Dandliker, and Williamson, 1976).
KeywordsFractional Order Association Constant Fluorescence Polarization Forward Rate Ionic Medium
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