Fluorescence anisotropy decay: finding the correct physical model

When the fluorescence intensity and fluorescence anisotropy decays can be described as sums of exponentials, a simplifying assumption is often made: each intensity decay lifetime associates with each rotational correlation time. Numerous biological systems exist where this assumption is invalid. We have been evaluating a general kinetic scheme applicable to all possible associations between lifetimes and rotational correlation times. For the simple case of two lifetimes and two rotational correlation times, nine association models exist. We have been testing the ability of these different association models to discriminate against one another. Using a Monte Carlo algorithm, synthetic anisotropy data sets were generated according to each association model. Each data set was then analyzed by all models. To deduce which association model was used to generate a data set, we found that a global analysis of a family of anisotropy data sets differing in an independent parameter(s) is required; an example would be variable intensity decay amplitudes from decays collected at several emission wavelengths. Anisotropy decays of the two tryptophans per subunit of liver alcohol dehydrogenase were also analyzed by all of the two-lifetime, two-correlation-time association models to determine if one or both tryptophans experience local depolarizing motions.