Antigen-induced conformational changes in antibodies and their Fab fragments studied by circular polarization of fluorescence.
Conformational changes induced in antibody molecules and in their Fab fragments by binding of antigen were investigated by the circular polarization of the fluorescence emitted by the tryptophan residues. This property of the fluorescence is related to the asymmetry, and thus to the conformation and environment, of the emitting chromophore. Changes in the circular polarization of the fluorescence of the antibody were observed upon binding of RNase to anti-RNase, of poly(DL-alanyl)-poly(L-lysine) to antipoly(D-alanine), and of the "loop" of lysozyme, a monovalent antigenic determinant, to anti"loop." The spectral changes were observed at different antigen-antibody ratios, including high antigen excess, indicating that they are due to antigen binding and not to aggregation. The circular polarization of fluorescence also detects changes in conformation of the different Fab fragments upon binding of the corresponding antigens. These changes in conformation were, however, markedly different from those observed for the whole antibody molecules, and indicated an interaction between the Fc and Fab fragments in the antibody molecule, and probably a change in the conformation of Fc upon binding of antigen to the antibody. In contrast, the small hapten, phosphorylcholine, did not induce a change in the circular polarization of the fluorescence of its antibody or corresponding Fab fragments. Reduction of the interchain disulfide bonds of the antibodies abolished the antigen-induced spectral changes due to the presence of the Fc portion in the molecule, but not the changes observed in Fab, suggesting that the disulfide bonds at the hinge region of the antibody are required for the transmission of the conformational change from the Fab to the Fc.
D000818:Animals; D000906:Antibodies; D000937:Antigen-Antibody Reactions; D000941:Antigens; D001665:Binding Sites; D001666:Binding Sites, Antibody; D002846:Chromatography, Affinity; D004220:Disulfides / Q000032:analysis; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D009113:Muramidase / Q000276:immunology; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits / Q000276:immunology; D012260:Ribonucleases / Q000276:immunology; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis
D000906:Antibodies; D000941:Antigens; D004220:Disulfides; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D012260:Ribonucleases; D009113:Muramidase