The conformational stability of B cell epitopes on the 25-kDa group I and 14-kDa group II mite allergens was compared by using heat-treated or chemically denatured allergens to inhibit the binding of native 125I allergens to murine mAb or to human IgE antibodies. Structural changes after treatment were assessed by SDS-PAGE and circular dichroism spectroscopy. Heating for 1 h at greater than 75 degrees C, treatment at pH 2.0 or pH 12.0, or with 6M guanidine or 6M urea, reduced the binding of the

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... group I allergens to mAb or IgE antibodies by 10- to 1000-fold. The group II allergens were heat stable and even after prolonged heat treatment (5 h at 75 degrees C or 30 min at 100 degrees C) their antibody binding activity was reduced by less than twofold. The group II allergens were also resistant to pH and to denaturation with 6M guanidine. However, after reduction and alkylation, antibody binding sites on both the group I and group II allergens were destroyed. Reduction of disulfide bonds with 2-ME caused a marked shift in the molecular mass of group I allergens on SDS-PAGE, from 25 kDa to 28-31 kDa. Reduction and alkylation also generated two high m.w. forms of Der p I and Der f I. After heating (100 degrees for 30 min), both Der f I and Der f II retained significant secondary structure, as judged by circular dichroism spectroscopy, but on reduction they showed the typical spectra of fully denatured proteins (greater than 85% random structure). The results show clear differences between the susceptibility of B cell epitopes on the group I and group II allergens to denaturation. Despite these differences in stability, both allergens are equally potent immunogens for IgE antibody responses in man. The results support the view that the physical properties of allergens (low m.w. and solubility), limiting low dose exposure (1 to 10 ng/day), and host genetic and immunoregulatory processes, are more important than gross structural features in the induction and maintenance of IgE antibody responses.