Measurements of fluorescent anisotropy decline of gamma-globulin and its fragments Fab, Fc, F(ab) 2 labelled with 1-sulfonyl-5-dimethyl-aminonaphthalene

Abstract

The time course of the two polarized components of the fluorescence excited by a very short flash has been used to determine the total fluorescence and the fluorescence anisotropy decays of dansylated γ-globulin, IgG and its enzymic fragments [Fab, Fc, F(ab)2]. The decays of the total fluorescence can be accounted for by the sums of three exponentials. The anisotropy decay curves can be interpreted by two relaxation processes. The first process, characterized by the relaxation θC, is very short and corresponds to a local motion of small fragments of the molecules. The second process, of relaxation time θL, is long and is due to a global motion of the molecules. The influence of θL is predominant in the final part of the decay curves. This part of the curve concerning Fab and Fc fragments can be interpreted by the model of the equivalent ellipsoid of about a similar size as that found in low angle X-ray scattering measurements. The analysis of the anisotropy decays of IgG and of F(ab)2 do not reveal a great freedom of rotation in the joint portion of the F(ab)2 and Fc fragments. Our results do not allow us to confirm the presence of some limited freedom of rotation in that part of the molecule, as has been proposed by Yguerabide, Epstein and Stryer [10]. Finally, we discuss the significance of the classical method of fluorescence depolarization where one uses a continuous excitation. It is shown that the slope of the depolarization curve is influenced by the variation of θC in the whole experimental range of T/η. Consequently, it is not possible to obtain directly the macromolecular relaxation time θL of the molecules of IgG and its fragments, using this method.