title: Perturbation of the antigen-binding site and staphylococcal protein A-binding site of IgG before significant changes in global conformation during denaturation: an equilibrium study.
abstract: Although conformational perturbation of the active sites of many enzymes has been reported to precede global molecular conformational changes [Tsou (1993) Science 262, 380-381], little effort has been made to compare the susceptibility of the ligand-binding site of proteins and the protein molecules as a whole to perturbation by denaturants. Immunoglobulin is chosen in this study to address this problem. It is found that the variable and constant regions (Fv and Fc) of a monoclonal antibody of an IgG subclass against adenylate kinase lose their abilities to bind antigen and staphylococcal Protein A after treatment with guanidinium chloride concentrations considerably lower than those required to change the global conformation of the antibody as a whole, as detected by fluorescence and second-derivative UV absorption spectroscopy. These results indicate that both ligand-binding sites of the antibody concerned are more fragile than the molecule as a whole and that the Fv and Fc regions of the antibody molecule unfold sequentially during denaturation.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, China.
authors.forename: X D
authors.initials: XD
authors.lastname: Wang
authors.forename: J
authors.initials: J
authors.lastname: Luo
authors.forename: Z Q
authors.initials: ZQ
authors.lastname: Guo
authors.forename: J M
authors.initials: JM
authors.lastname: Zhou
authors.forename: C L
authors.initials: CL
authors.lastname: Tsou
mesh_terms: D000941:Antigens / Q000378:metabolism*; D001665:Binding Sites; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013205:Staphylococcal Protein A / Q000378:metabolism*
chemical_list: D000941:Antigens; D006146:Guanidines; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; D019791:Guanidine