Mn2+-probe ESR method for the analyses of the dissociation of charged residues on the surface of immunoglobulins.
Dissociation of charged residues on the surface of immunoglobulins was analysed by an Mn2+ probe ESR method that has been developed in our previous work. Several kinds of IgG proteins and their Fab and Fc fragments were used for the experiments. The pH dependence of the intensity of ESR signals was analysed. It was shown that the number of Asp, Glu and His residues on the surface of Fc is about twice as many as that of Fab. The accessible surface area of amino acid residues calculated using X-ray crystallographic data is quite consistent with the present ESR experiments. This indicates that the number of the Asp, Glu and His residues on the surface of IgG molecules in solution is similar to that in the crystal. The Mn2+-probe ESR method was also applied to other classes of immunoglobulins, i.e. IgA and IgM. It was demonstrated that the IgA protein, which is known to lack the ability to bind Clq, has on the surface of it a smaller number of Asp, Glu and His residues as compared to IgG and IgM proteins. On the basis of these results obtained by the Mn2+-probe ESR method, we suggest that the Clq molecule, which is a basic protein, interacts favorably with the Fc portion whose surface is more negatively charged with Asp and Glu residues, compared to the Fab portion. Fine adjustment of fitting of the head of the Clq molecule into the CH2 domain of the Fc portion presumably follows for optimum binding. It was also demonstrated that Ser and Thr residues are much more abundant on the surface of Fab than in the case of Fc. We suggest that the Ser and Thr residues on the surface of Fab play an important role for binding of C4b upon activation of the complement system.
D001216:Asparagine / Q000378:metabolism; D001666:Binding Sites, Antibody; D003461:Crystallography; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005973:Glutamine / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007070:Immunoglobulin A / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D007075:Immunoglobulin M / Q000378:metabolism; D007136:Immunoglobulins / Q000378:metabolism*; D008345:Manganese; D009194:Myeloma Proteins / Q000378:metabolism
D007070:Immunoglobulin A; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007136:Immunoglobulins; D009194:Myeloma Proteins; D005973:Glutamine; D008345:Manganese; D006639:Histidine; D001216:Asparagine