PERTURBATION OF THE ANTIGEN-BINDING SITE AND STAPHYLOCOCCAL PROTEIN A-BINDING SITE OF IGG BEFORE SIGNIFICANT CHANGES IN GLOBAL CONFORMATIONDURING DENATURATION - AN EQUILIBRIUM STUDY

Although conformational perturbation of the active sites of many enzym es has been reported to precede global molecular conformational change s [Tsou (1993) Science 262, 380-381], little effort has been made to c ompare the susceptibility of the ligand-binding site of proteins and t he protein molecules as a whole to perturbation by denaturants. Immuno globulin is chosen in this study to address this problem. It is found that the variable and constant regions (Fv and Fc) of a monoclonal ant ibody of an IgG subclass against adenylate kinase lose their abilities to bind antigen and staphylococcal Protein A after treatment with gua nidinium chloride concentrations considerably lower than those require d to change the global conformation of the antibody as a whole, as det ected by fluorescence and second-derivative UV absorption spectroscopy . These results indicate that both ligand-binding sites of the antibod y 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.