Formation of Biologically Inactive Polymers is Responsible for the Thermal Inactivation of Rat IgE

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Abstract
The structural changes induced by heating rat IgE at 56 °C and relationship with loss of cytotropic activity were examinated in the present study. Circular dichroism spectrum of IgE heated at 56 °C showed irreversible changes in the peptide bond spectral regions: increase in β-sheet structure, but no significant modifications in the aromatic side chain region. Thus, circular dichroism studies did not suggest important perturbations of the tertiary structure of the IgE molecule. Parallel studies with F(ab’)2-ε fragment did not show significant alterations of either peptide bond or aromatic side chain spectral regions. Analysis of IgE heated at 56 °C by polyacrylamide gradient gel electrophoresis showed the presence of large amounts of polymeric material. Polymerization of IgE was found to increase with time of heating at 56 °C and to depend on protein concentration; polymerization was decreased at temperatures lower than 56 °C. A relationship between loss of cytotropic activity and the proportion of polymeric material in the heated IgE solutions was observed. Isolated polymeric molecules produced by heating showed considerable decrease in cytotropic activity whereas monomer isolated from heated IgE was found biologically active. The ability to form polymers is an intrinsic property of the carboxy-terminal domains Cε3 and Cε4, as the F(ab′)2-ε fragment did not polymerize upon heating at 56°C. A model of thermal inactivation of rat IgE is proposed in which aggregation of the carboxy-terminal domains of the ε-chain does not allow interaction of these domains with the monovalent IgE receptor of mast cells.