Determination of rotational correlation times from deconvoluted fluorescence anisotropy decay curves. Demonstration with 6,7-dimethyl-8-ribityllumazine and lumazine protein from Photobacterium leiognathi as fluorescent indicators

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Abstract
The experimental and analytical protocols required for obtaining rotational correlation times of biological macromolecules from fluorescence anisotropy decay measurements are described. As an example, the lumazine protein from P. leiognathi was used. This stable protein (MW 21,200) contains the nonconvalently bound, natural fluorescent marker 6.7-dimethyl-8-ribityllumazine, which has in the bound state a long fluorescence lifetime (.tau. = 14 ns). Shortening of the fluorescence lifetime to 2.6 ns at room temperature was achieved by addition of the collisional fluorescence quencher potassium iodide. The shortening of .tau. had virtually no effect on the rotational correlation time of the lumazine protein (.vphi. = 9.4 ns, 19.degree. C). The ability to measure biexponential anisotropy decay was tested by the addition of Photobacterium luciferase (MW 80,000), which forms an equilibrium complex with lumazine protein. Under the experimental conditions used (2.degree. C) the biexponential anisotropy decay can best be described with correlation times of 20 and 60 ns, representing the uncomplexed and luciferase-associated lumazine proteins, respectively. The unbound 6,7-dimethyl-8-ribityllumazine itself (.tau. = 9 ns) was used as a model compound for determing correlation times in the ps time range. In the latter case rigorous deconvolution from the excitation profile was required to recover the correlation time, which was shorter (100-200 ps) than the measured laser excitation pulse width (500 ps).