A general method for constrained analysis of fluorescence anisotropy decay: Application of the steady-state anisotropy

Time-resolved fluorescence anisotropy is an invaluable method for investiga ting the internal and rotational dynamics of biomolecules. The range of rot ational motions detectable by anisotropy decay is limited by the fluorescen ce Lifetime; typically, a depolarizing motion may be resolved if the associ ated correlation time is between 0.1 and 10 times the intensity decay lifet ime. To extend that range and to improve the recovery of anisotropy decay p arameters, a general analytical method has been developed. This procedure u tilizes a modification of Lagrange multiplier methods to constrain the valu es of the iterated kinetic parameters during nonlinear least-squares analys is of anisotropy decay data. The form of the constraint equation is derived from the classic relationship between the decay parameters and the steady- state anisotropy, which can be simply and accurately measured. Application of the constraint to analyses of synthetic data sets increased the accuracy of recovery by decreasing the uncertainty in the iterated parameters. The constraint also enabled the accurate recovery of correlation times that wer e a factor of 30 greater than the fluorescence lifetime, although it did no t improve recovery of correlation times that were much shorter than the lif etime. Using this technique, it should now be possible to characterize the dynamics of larger macromolecules and assemblies than those that can curren tly be studied by fluorescence anisotropy decay.