Proximity relationships within the Fc segment of rabbit immunoglobulin G analyzed by resonance energy transfer.
Resonance energy transfer analysis has been carried out with a noncovalent rabbit hybrid of immunoglobulin G (IgG) composed of normal rabbit IgG and rabbit anti-lactose IgG. The hybrid IgG was prepared from proteins in which the single inter-heavy-chain disulfide linkage was specifically reduced and alkylated. Normal rabbit IgG was alkylated with the iodoacetyl derivative of N-(aminoethyl)-5-naphthylamine-1-sulfonic acid while the rabbit anti-lactose IgG was alkylated with either iodoacetamide or the iodoacetyl derivative of p-[[p-(dimethylamino)phenyl]azo]aniline. Fractionation with an anti-lactose-specific immunoadsorbent yielded a population in which each fluorescent donor [N-[(acetylamino)ethyl]-5-naphthylamine-1-sulfonic acid] was adjacent to a nonfluorescent acceptor [N-acetyl-p[[p-dimethylamino)phenyl]. Data on fluorescence quantum yield, excited-state lifetime, time-resolved emission anisotropy, and steady-state fluorescence polarization revealed a distribution of distances between the donor and acceptor. In the native molecule, the hinge regions are known to be covalently liked by a single disulfide bond. In the absence of this linkage, the hinge regions were separated such that for a majority of the molecules in solution (approximately 60%) the separation was 50-60 A. In the context of current knowledge of the IgG molecule, it is evident that the principle forces maintaining the integrity of the native, functional Fc segment are the strong noncovalent interactions of the CH3 domains and the single inter-heavy-chain disulfide bond.
D000818:Animals; D004735:Energy Transfer; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007164:Immunosorbents; D007700:Kinetics; D008433:Mathematics; D011487:Protein Conformation; D055503:Protein Multimerization; D011789:Quantum Theory; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007164:Immunosorbents