Time-Dependent Anisotropy Decays

Abstract

In the preceding chapter we described the measurement and interpretation of steady-state fluorescence anisotropies. These values are measured using continuous illumination, and represent an average of the anisotropy decay by the intensity decay. The measurement of steady-state anisotro-pies is simple. However, interpretation of the steady-state anisotropies usually depends on an assumed form for the anisotropy decay, which is not observed in the experiment. Additional information is available from measurements of the time-dependent anisotropy, that is, the values of r(t) following pulsed excitation. The form of the anisotropy decay depends on the size, shape, and flexibility of the labeled molecule. The measured decays can be compared with the decays calculated from various molecular models. Ani-sotropies decays can be measured using the time-domain (TD) or the frequency-domain (FD) method. It is important to understand the factors which affect the anisotropy decays. For a spherical molecule the anisot-ropy is expected to decay with a single rotational correlation time (?). Perhaps the most frequent interpretation of the correlation time is in terms of the overall rotational correlation time of a protein. The measured values of ? can be compared with the values predicted for a hydrated sphere of equivalent molecular weight (eq. 10.46). However, ani-sotropy decays are usually multi-exponential, which can be the result of numerous factors. Multi-exponential anisot-ropy decays are expected for non-spherical fluorophores or proteins. The correlation times in the anisotropy decay are determined by the rates of rotation about the various molecular axes. By examination of the correlation time it is sometimes possible to estimate the shapes of proteins.