Interactions of IgG with specific erythrocyte membrane proteins in affinity electrophoresis are highly dependent on low ionic strength conditions.
Agarose gel affinity electrophoresis has been used to demonstrate interactions between autologous IgG and specific erythrocyte membrane proteins. These binding phenomena are here further examined by combining affinity electrophoresis with affinity chromatography, absorption experiments, and immunoblotting. It is demonstrated that the interactions are highly dependent on polyreactive IgG binding favored by the low ionic strength conditions of the electrophoretic assay. Thus, about 25% of normal IgG under low ionic strength conditions bound to the purified cytoskeletal protein, spectrin, immobilized on Sepharose. This IgG reacted in affinity electrophoresis in a polyspecific fashion with the same array of membrane proteins as before the low ionic strength-affinity chromatography. Further, the binding seen in affinity electrophoresis, including the interaction with spectrin, was completely abolished by preabsorption of the IgG with spectrin-devoid membranes. The charge characteristics of an IgG subclass might be responsible for the observed binding. However, the observed precipitate formation suggested an interaction involving at least two binding sites on each molecule and the binding appears to require structurally intact IgG because reductive treatment with dithiothreitol diminished the reactivity considerably. Conclusively, under the conditions of affinity electrophoresis with ligand present in the gel, electrostatic interactions are amplified. The degree of binding of IgG to erythrocyte membrane proteins that take place under these conditions does not reflect binding which would occur to the same extent under physiological ionic strength conditions.
Heegaard
N H
NH
Laboratory of Biochemical Genetics, NIMH Neuroscience Center, Saint Elizabeth's Hospital, Washington, DC.
D001792:Blood Platelets / Q000378:metabolism; D001797:Blood Protein Electrophoresis / Q000379:methods*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005069:Evaluation Studies as Topic; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000097:blood* / Q000302:isolation & purification; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011485:Protein Binding
D007074:Immunoglobulin G; D008565:Membrane Proteins