Separation of complexes containing protein A and IgG or Fc gamma fragments by high-performance liquid chromatography

Protein A of Staphylococcus aureus is a bivalent Fc receptor that can form complexes with immunoglobulin G (IgG) or Fc gamma fragments that activate humoral (e.g., complement) and cellular (e.g., lymphocyte) components of the immune system both in vitro and in vivo. To obtain complexes formed between protein A of Staphylococcus aureus (SpA) and rabbit IgG or Fc gamma fragments for purposes of characterizing their compositions and studying their biological activities, we have used high-performance liquid chromatography to separate complexes in 20 min. Complexes were prepared with trace amounts of 125I-SpA and 131I-IgG or 131I-Fc gamma to simplify the analyses. With excess molar amounts of IgG or Fc gamma the complexes have the molecular formulas [(IgG)2SpA]2 or [(Fc gamma)2SpA]2. With excess SpA, complexes corresponding to (IgG)(SpA) or (Fc gamma)(SpA) are formed, perhaps with other complexes that have different ratios of components. Since SpA is a rod-shaped molecule it elutes at a molecular weight corresponding to 240,000 rather than the true value of 42,000. This behavior is reflected in the elution of certain complexes at shorter retention times than expected on the basis of actual molecular weights, and facilitates separation of complexes from free IgG or Fc gamma. The true molecular weights and molecular formulas of complexes isolated by HPLC were verified by ultracentrifugation. This HPLC method was used to study the interconversion and stability of complexes.