DYNAMICS OF BIOMOLECULES - ASSIGNMENT OF LOCAL MOTIONS BY FLUORESCENCE ANISOTROPY DECAY

Many biological systems have multiple fluorophores that experience mul tiple depolarizing motions, requiring multiple lifetimes and correlati on times to define the fluorescence intensity and anisotropy decays, r espectively. To simplify analyses, an assumption often made is that al l fluorophores experience all depolarizing motions. However, this assu mption usually is invalid, because each lifetime is not necessarily as sociated with each correlation time. To help establish the correct ass ociations and recover accurate kinetic parameters, a general kinetic s cheme that can examine all possible associations is presented. Using s ynthetic data sets, the ability of the scheme to discriminate among al l nine association models possible for two lifetimes and two correlati on times has been evaluated. Correct determination of the association model, and accurate recovery of the decay parameters, required the glo bal analysis of related data sets. This general kinetic scheme was the n used for global analyses of liver alcohol dehydrogenase anisotropy d ata sets. The results indicate that only one of the two tryptophan res idues in each subunit is depolarized by process(es) independent of the enzyme's rotations. By applying the proper kinetic scheme and appropr iate analysis procedures to time-resolved fluorescence anisotropy data , it is therefore possible to examine the dynamics of specific portion s of a macromolecule in solution.