Effects of ligand binding upon measurement of Gc by rocket immunoelectrophoresis: Implications for protein determination and for studies of protein/ligand interactions

The effect of protein complexing on quantitation by rocket immunoelectrophoresis was studied using group‐specific component (Gc), a serum protein which undergoes 1:1 interactions with both sterols (Vitamin D metabolites) and protein (G‐actin). Gc purified from serum was used as a reference. Complex formation with 25‐〈OH〉 D3 did not significantly affect the measurement of Gc. However, addition of excess G‐actin to purified Gc led to substantially higher rockets at every concentration of Gc tested. In dose‐response experiments performed with a constant quantity of Gc and increasing quantities of added G‐actin, the increment in rocket height was progressive up to equimolarity, at which point a plateau was reached. Further demonstration that the increased rocket heights were directly related to 1:1 complex formation between Gc and G‐actin (Gc:G‐actin) was obtained by [¹²⁵I]G‐acting and isoelectric focusing (IEF) with autoradiography. Examination of the relative charge of Gc and of Gc:G‐actin by crossed immunoelectrophoresis showed that complexes exhibited faster mobility, whereas very little evidence of alteration in antigenicity of Gc:G‐actin complexes was apparent. These results suggest that Gc:G‐actin complexes exhibit increased electrophoretic mobility and that their presence in samples containing Gc causes an increase in the height of Gc rockets. The findings also indicate that interactions with specific ligands could cause artefacts in immunoelectrophorestic quantitation of other proteins present in biological fluids.