The role of the thiol group and the disulfide bond in the conformation and the IgE and IgG binding activities of ragweed allergen Ra3

Abstract

A single free thiol group was alkylated by reacting allergen Ra3 with iodoacetamide or iodoacetic acid. Reduction with mercaptoethanol followed by carboxymethylation resulted in the reduction and alkylation of a single disulfide bond, in addition to the alkylation of the free thiol group. CD § measurements on Ra3 and its derivatives indicated that carboxyalkylation of the single free SH group by either iodoacetamide or iodoacetic acid caused slight conformational changes in Ra3. On the other hand, the conformational changes resulting from cleavage of the disulfide bond were quite large and the CD spectra reflected transition to a state of largely unordered structure. The immunochemical reactivities of the derivatives with anti-Ra3 human IgE and IgG antibodies as well as rabbit IgG antibodies were determined by direct binding of the respective antibodies to the adsorbents of the derivatives and/or by inhibition of the binding between the antibodies and native Ra3 adsorbents by the various derivatives in solution. Modification of the single thiol group caused only a very slight decrease (about 5%) of the reactivities with anti-Ra3 human IgE antibodies or with IgG antibodies of human or rabbit origin. Reduction followed by carboxyalkylation of all three thiol groups decreased the reactivity with IgE or IgG antibodies by about 50%. The behavior of a given derivative with the three antibodies was virtually the same. It was concluded that the free thiol group is not an essential part of an allergenic (IgE binding) or antigenic (IgG binding) site in Ra3. On the other hand, the intactness of the disulfide bond is essential for maintaining the integrity of the native conformation of Ra3 and the binding capacity of about half its allergenic and antigenic sites.