Electron microscopic and hydrodynamic studies of protein A-immunoglobulin G soluble complexes

The soluble complexes formed by reacting staphylococcal protein A (SpA) with rabbit immunoglobulin G (IgG) antibodies were characterized by hydrodynamic and electron microscopic methods. In moderate SpA excess, equilibrium mixtures of SpA and rabbit IgG formed four discrete complexes that sedimented at approximately 7, 10, 13, and 15S. The putative complexes were visible by electron microscopy and appeared to contain one, two, three, and approximately five molecules of IgG. Probably because of its elongated shape, SpA was not clearly visible in these mixtures or in control preparations of SpA alone. Both native IgG and IgG modified by cleavage of its single-hinge disulfide bond formed similar complexes on interaction with SpA. It was possible to resolve heterogeneous mixtures of IgG-SpA complexes by using an analytical ultracentrifuge equipped with a photoelectric scanner interfaced to a small computer. The relative concentrations and sedimentation velocities of different complexes in a mixture were determined from computer-generated integral and derivative plots. Both hydrodynamic and electron microscopic methods revealed that the distribution of complexes was sensitive to the IgG to SpA molar ratio. The relative amounts of faster complexes increased as the IgG to SpA molar ratio was increased. Surprisingly, when the IgG to SpA molar ratio was greater than or equal to 2, the complexes were converted into a unique 17S complex. This rather unprecedented transformation was reversible: the addition of excess SpA caused the dissociation of the 17S complex into a mixture of the 7, 10, 13, and 15S structures. The average translational diffusion coefficient of the 17S complex was 2.62 +/- 0.13 Ficks. In the electron microscope, the complex appeared to be exceptionally compact with an average diameter of 287 A. The stoichiometry of the 17S complex, together with sedimentation equilibrium, diffusion, and electron microscopic measurements, indicated that it is composed of four molecules of IgG and two molecules of SpA.