Allergens of Alternaria: further characterization of a basic allergen fraction

A basic allergen fraction isolated from Alternaria extracts by preparative flat-bed gel isoelectric focusing (as previously reported) was subjected to further characterization. The fraction was chromatographed on a Sephadex G-25 column and exhibited two major peaks, the first of which was allergenic and the second of which was nonallergenic but caused a precipitate to form when added to a mixture of sodium dodecyl sulfate (SDS) (1%) and trichloroacetic acid (5%). SDS-PAGE indicated that the allergen component of the basic fraction migrated at about the same rate as insulin (5.8 kdaltons). Both G-25 peaks were carbohydrate rich with a protein:carbohydrate ratio (by weight) of 1:4.5 and 1:5.8 for peak 1 and peak 2 respectively. Heat (100 degrees C for 10 min) and enzyme digestion (trypsin, alpha-chymotrypsin and pepsin) did not reduce the RAST inhibition values for peak 1 or the original basic fraction. The basic fraction did not bind human IgE specific for birch pollen indicating that the RAST inhibition and IgE (Alternaria specific) binding to radioimmunoelectrophoresis plates was not due to nonspecific binding of IgE. We conclude that the allergenicity of the basic fraction is due to a small molecule that is predominantly carbohydrate in nature and is a major allergen of Alternaria.