Comparative Studies on Tree-Pollen Allergens

Gel permeation chromatography of Betula verrucosa (BV) crude pollenextract and pooling of the fractions after their fused rocket immunoelectrophoresis antigenic profile allowed the isolation of a purified major allergen designated BV45. This fraction was shown to be a glycoprotein with a tentative molecular weight of 10,000 daltons. The estimated molecular weight differed by approximately factor 2 from the previously reported values of birch pollen major allergens. This fraction was further purified by polyacrylamide gel chromatography (P-10), high performance liquid chomatography (HPLC) silica-base column and high-volt electrophoresis at different dimensions. The purified proteins revealed an immunochemical identity with the initial material. BV45 and the previously isolated pI 5.18, though different otherwise, demonstrated a reaction of complete antigenic identity illustrated by tandem-crossed immunoelectrophoresis. Both the amino acid and carbohydrate compositions of BV45 were calculated on the basis of the tentative molecular weight obtained. BV45 showed a total carbohydrate concentration of 20% of the dry weight, corresponding to 13–14 residues per mol protein. The amino acid composition obtained reflected structural diversity between the BV45 and pI 5.18. N-terminal analysis followed by HPLC quantitation of the DNS-amino acids for BV45 and the further purification products gave two peaks having retention times corresponding to DNS-Cya and DNS-Asp. Fraction BV45 was demonstrated to be a major allergen of birch pollen as assessed by RAST inhibition of 7 out of 10 reaginic sera and the specific radiostaining in crossed radioimmunoelectrophoresis of all omitt nine crossed immunoelectrophoresis plates used. RAST inhibition titration of BV45 and BV crude showed qualitative identity of the allergenic determinants of both materials. The question about one or several major allergen(s) in birch pollen extract has so far been unsolved as no structural mapping of BV45 and pI 5.18 has been readily available.