PURIFICATION AND CHARACTERIZATION OF A SOYBEAN HULL ALLERGEN RESPONSIBLE FOR THE BARCELONA ASTHMA OUTBREAKS .2. PURIFICATION AND SEQUENCINGOF THE GLY-M-2 ALLERGEN

Background A low MW allergen from soybean hull, Gly m 1, with two isoa llergens, Gly m 1 A and Gly m 1 B, was associated with the asthma outb reaks that occurred in Cartagena, Spain. Using sera of asthmatic epide mic patients (AEP) from Barcelona, three main soybean hull allergens, two of them with MWs and pIs identical to those reported for Gly m 1 A and Gly m 1 B, were identified. Objective The purpose of this study w as to purify and to study the N-terminal amino acid sequence of the th ird allergen, which has a MW of 8 kDa. Method The purification procedu re combined the double dialysis method and preparative isoelectofocusi ng (IEF). Specific IgE determination to the fractions obtained demonst rated three peaks, one of them corresponding to the 8 kDa allergen. Th e pooled fractions containing this allergen were studied by sodium dod ecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-PAGE/We stern blot and IEF/Western blot. Only a band with a MW of 8 kDa and a pI of 6 was obtained. Its allergenic activity was measured and it was demonstrated that the allergenicity of soybean hull correlates with th e presence of the 8 kDa allergen. The N-terminal amino acid sequence o f the first 20 amino acids, which was registered at the PIR Data Submi ssion as the N-terminal partial sequence of Gly m 2, was determined ac cording the Edman degradation method. Results Gly m 2 N-terminal amino acid sequence lacks homology with that reported for the allergen Gly m 1 but has a homology of 71% with a storage protein from cotyledon of Vignla radiata (cow pea) and 64% with a 'disease response protein' fr om Pisum sativum (green pea). These results suggest that Gly m 2 in so ybeans could protect against diseases which affect soybean plants. Con clusion This study demonstrates the existence of another soybean hull allergen, Gly m 2, partially responsible for the soybean asthma outbre aks that occurred in Barcelona, Spain.